Ultimate Protocols in Biotechnology and Molecular Biology

2013-12-10T14:11:00.000-08:00

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Particle Bombardment / Gene Gun

2010-07-23T22:42:00.000-07:00

Particle bombardment using biolistic gun

(The protocol is described as such it is performed in Plant Transformation Lab)

Preparation of DNA for PDS 1000/He gene gun:

This gun was originally constructed by Sanford et al (1992) and later marketed by BIO-RAD Company.

A. Micro-carrier preparation

1. In a 1.5ml microfuge tube, weigh out 3 mg of microparticles (gold particles 1.0 mm) for 6 bombardments.

2. Add 1ml of 70 % ethanol, fresh prepared.

3. Vortex on a platform vortexer for 2-3 minutes.

4. Incubate for 15 minutes.

5. Pellete the microparticles by spinning for 5 seconds in a microfuge.

6. Remove the liquid and discard.

7. Repeat the following steps three times.

a. Add 1ml of sterile water.

b. Vortex for 1 minute.

c. Allow the particle to settle for 1 minute.

d. Pellete the microparticles by spinning for 2 seconds in a microfuge.

e. Remove the liquid and discard.

8. Add 50 ml of sterile water to bring the microparticle concentration to 60 mg/ml.

9. Store the microparticles at room temperature for up to 2 weeks.

B. Coating DNA onto microparticles

The following procedure is sufficient for six bombardments; if less bombardment is needed, prepare enough microcarriers for three bombardments by reducing all volumes by one-half. When removing aliquots of microcarriers, it is important to vortex the tube containing the microcarriers continuously in order to maximize uniform sampling.

1. Vortex the micrcarriers prepared in sterile water (60 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles.

2. Remove 50 ml (3 mg) of microcarriers to a 1.5 ml microfuge tube.

3. While vortexing vigorously, add in order:

a. 20ul spermidine (0.1 M)

b. 50 ul CaCl₂ (2.5 M) was added drop by drop on vortex and continued for 2-3 minutes

4. Allow the microcarrier to settle for 1 minute.

5. Pellet the microcarriers by spinning for 2 seconds in a microfuge.

6. Remove the liquid and discard.

7. Add 140 ml of 70 % ethanol without disturbing the pellet.

8. Remove the liquid and discard.

9. Add 140 ml of 100 % ethanol without disturbing the pellet.

10. Remove the liquid and discard.

11. Add 48 ml of 100% ethanol.

12. Gently resuspend the pellet by tapping the side of the tube several times, and then by vortexing at low speed for 2-3 seconds.

13. Remove 6ul aliquots of microcarriers and transfer them to the center of a macrocarrier. An effort is made to remove equal amounts (500 mg) of microcarriers each time and to spread them evenly over the central 1 cm of the macrocarrier using the pipette tip and Desiccate immediately.

14. Fix macrocarrier, rupture disk and sieve on the instrument.

15. Build up 1100 psi pressure to the rupture disk and shoot the DNA on the cells of scueltellum callus.

16. Culture the tissue on the medium containing manitol (described above) to maintain the osmatic pressure.

Antibiotic Stock Solutions

2010-07-23T22:37:00.000-07:00

Antibiotics Stock Solutions (Practically performed working concentration stock solutions for Agrobacterium and E.coli))
Kanamycin (stock 100 mg/ml)

1. Kanamycin-sulphate (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Cefotaxime (Claforan) (stock 100 mg/ml)

1. Cefotaxime (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Ampicilin (stock 100 mg/ml)

1. Ampicilin (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Transformation / Electroporation of Agrobacterium tumefaciens

2010-07-23T03:33:00.000-07:00

Transformation of clones in Agrobacterium tumefaciens by electroporation

(The performed protocol using earlier described electro-competent cells of Agrobacterium tumefaciens)

Only 2 ml of each clone was used for electro-transformation by electro cell manipulator 600 (BTX San Diego, California). For electroporation the following protocol was used.

1. Electroporation cuvettes 1 mm gap were placed on ice. Viols of frozen electro-competent cells of Agrobacterium were allowed to thaw on ice.

2. 1 mg DNA of the recombinant plasmid was mixed with 50 ml of electro-competent cells in the electroporation cuvettes on ice.

3. The condition for electroporation were set as recommended by the manufacturer:

Choose mode T 2.5 KV

Set resistance R R5 (129ohm)

Set charging voltage 1.44 KV

4. The electro-competent cells containing the DNA mixture were transferred to electroporation cuvette.

5. Pulse was given and 1 ml of liquid LB medium was added immediately, mixed gently and transferred to a 1.5 ml eppendorf tube and incubated at 28°C for 1 hour with vigorous shaking.

6. 200 μl and 400 μl of transformed culture were spread on petri plates containing solid LB medium supplemented with 50 µg of rifampicin and 50 µg of kanamycin/ml, so that only transformed cells should multiply.

7. When the liquid was absorbed completely the plates were sealed with sealing film and kept at 28 ⁰C for 2-3 days.

8. At the end of incubation colonies were picked with sterile toothpicks and cultured in 5ml liquid LB medium in 50 ml tube containing 50 µg of rifampicin and 50 µg of kanamycin/ml.

9. Culture tubes were kept at 28 ⁰C on shaker in Agrobacterium growth room for 48 hours with vigorous shaking.

Transformants were confirmed through PCR.

Preparation of Agrobacterium Electro-Competent Cells

2010-07-23T03:28:00.000-07:00

Preparation of electro-competent cells of Agrobacterium tumefaciens

1. Pick a single colony from a freshly grown plate of Agrobacterium and inoculate into 100 ml LB liquid medium in 250 ml autoclaved flask using sterile toothpick and incubate at 28 ⁰C for 48 hours with vigorous shaking.

2. Add 5 ml of the 48 hours grown culture re-inoculate into 1 liter flask containing 250 ml of the same LB medium and incubate at 28 ⁰C untill OD₆₀₀ of cells become 0.5-1.0 (10¹⁰ cells/ml).

3. Transfer the cells aseptically to ice cold 50 ml propylene tube and keep cool on ice for 30 minutes.

4. Now centrifuge the cells at 4000 rpm for 10 minutes in a bench top refrigerated centrifuge machine at 4 ⁰C. Decant the supernatant and re-suspend the pelleted in 50 ml of sterile cold ddH₂O.

5. Again centrifuge the cells at 4000 rpm in the same centrifuge at 4 ⁰C for 10 minutes and decant the supernatant. Again re-suspend the pelleted cells in 25 ml of sterile cold ddH₂O.

6. After another wash, re-suspend the cells in 10 ml sterile cold ddH₂O containing filter sterilized cold 10% glycerol. This wash should be repeated.

Finally re-suspend the the cells in 1-1.5 ml filter sterilized cold 10% glycerol, aliquote in 50 ml and store at -70 ^oC.

Transformation of E.coli Cells

2010-07-23T00:27:00.000-07:00

Transformation in E. coli DH5- α by electroporation

For electroporation following protocol can be used.

1. Electroporation cuvetts 2 mm gap place on ice.

2. Vials of frozen electrocompetent cells of DH5-α allow to thaw on ice.

3. 2 μl of ligation mixtures pipette into Eppendorf tube containing the competent cells and mixe gently with the pipette tip.

4. The conditions for electroporation are:

Choose mode T 2.5 KV

Set resistance R R5 (129 ohm)

Chamber gap 2 mm

Set charging voltage 2.45 KV

5. The electrocompetent cells containing the ligation mixture then transfer to

electroporation cuvette.

6. Pulse the mixture and add 1 ml of liquid LB medium immediately, mix gently and transfer to a 1.5 ml Eppendorf tube and incubate at 37 ⁰C for 45 minutes with vigorous shaking.

7. Spread 100 μl of transformed culture on solid LB medium having antibiotic.

8. After the liquid absorbed completely, seal the plates with sealing film and keep at 37 ⁰C in incubation for over night.

9. Pick the Colonies with sterile toothpicks and culture in 3 ml liquid L.B medium containing relevant antibiotic.

10. Keep culture tubes at 37 ⁰C in water bath for over night with vigorous shaking.

11. Isolate plasmid and checked on 1% agarose gel.

Preparation of E.Coli Competent Cells

2010-07-23T00:19:00.000-07:00

Preparation of electro-competent cells of E. coli DH5-α

1. A single colony from a freshly grown plate of DH5-α(GIBCO-BRL, USA) was picked and transferred into 100 ml LB medium in 1 liter flask and incubated at 37 ⁰C overnight with vigorous shaking.

2. 2.5 ml of the overnight culture was taken and transferred to 250 ml LB in 1000 ml flask and shaken vigorously at 37 ⁰C until O. D⁶⁰⁰ of 0.5-1.0 (10¹⁰ cells/ml).

3. The cells were transferred aseptically to sterile disposable 50 ml propylene tube.

4. The culture was cooled by keeping on ice for 10 minutes.

5. The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in one of volume sterile cold distilled water.

6. The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.5 volume of sterile cold distilled water.

7. The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.02 volume of sterile cold distilled water.

8. The cells were again pelleted by centrifugation at 5000 rpm for 15 minutes and suspended finally in 0.002-0.003 volume sterile 10% cold glycerol.

9. The cells were stored in aliquots of 50 μl or 100 μl at -70 ⁰C.

Viruses Resistance Strategies in Plants

2009-08-25T09:11:00.000-07:00

Review

Viruses Resistance Strategies in plants

*Nabeel Akhtar, Amir Abdual Hakeem, Asad Ullah Khan

Institute of Biotechnology, B.Z University, Multan, Pakistan ============================================================

Abstract

Globally, virus diseases are common in agricultural crops and have a major agronomic impact. Virus infections cause physiological changes, symptoms and eventual yield losses in plants. They are countered through the Genetic engineering. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as ‘coat-protein-mediated’ protection. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. An other approach which was used recently is micro RNA. It (miRNA) is 21 nucleotide long sequences that regulate the abundance of target mRNA by guiding cleavage at sequence complementary. The sequence in pre mi RNA can be modified to produce artificial miRNA (amiRNA) of pre determined sequence. Further strategies for engineered virus resistance have been explored, including the use of pokeweed antiviral protein, oligoadenylate synthetase and ‘‘plantibodies’’. So here we make review of above discussed strategies.

INTRODUCTION

At present, plant viral diseases cause significant losses in crop quantity and quality. Naturally-occurring virus resistance genes have been introduced into commercial crop varieties by traditional plant breeding techniques. This is a proven approach but is limited by the availability of such genes and the many years of effort that this approach requires. Genetic engineering has become a successful strategy for producing virus resistant crop plants since 1986. Several candidate genes from naturally resistant plants are now becoming available with which to produce transgenic plants. However, the most common approach to obtain resistance does not use resistance genes. Current commercial transgenic crop plants, such as NatureMark's New Leaf Y potato, express resistance to viral infection by inserting a gene that codes for a protein on the outside of the virus called the "coat protein". This mechanism of developing resistance to viral infection by expressing only a single viral protein is referred to as "pathogen-derived resistance." A wide assortment of viral genes has been demonstrated to confer resistance against infection by that virus. This observation suggests that initiation of plant viral disease can be disrupted by a number of different processes. However, the precise mechanisms involved in disease resistance by transgenic plants are unknown. The use of genetic engineering to produce plants which are resistant to viral diseases has proven effective and is likely to be deployed widely in agriculture. For this reason, it is important to determine whether large-scale deployment of transgenic plants containing and expressing viral genes is safe for the environment, agriculture and human health. Increased knowledge of both the molecular genetics of plant viruses and, more recently, also of their hosts’ natural defence systems have resulted in the development of a number of novel ways to control virus diseases in plants. This paper presents a brief overview of novel insights in the natural defense mechanisms of plants to viruses as

Natural resistance to plant viruses

Besides passive defense based on the presence of existing barriers like the rigid cell wall, plants exhibit active defiance mechanisms upon recognition of pathogens such as viruses. The most common mechanism associated with active defense is the so-called hypersensitive response (HR), during which cells surrounding the primary infection site of the virus die due to a rapidly induced programmed cell death, which results in a visible necrotic local lesion. The induction of this response is preceded by a specific recognition of the virus, and in many cases this is based on matching (dominant) gene products of the plant (produced from dominant resistance genes, R genes) and the virus (avirulence genes). To date a few dozen single dominant R resistance genes recognizing different categories of plant pathogens have been cloned and sequenced.

Table 1

Selected set of dominant resistance (R) genes against viruses and other pathogens

R gene

Plant

Pathogen

Type

Prf

HRT

Sw-5

12c

Bs2

Mla 1

RPS4

Cf9

Tomato

Potato

Arabidopsis

Tomato

Pepper

Barley

Tobacco

Arabidopsis

Tomato

P. syringae

Meloidogyne incognita

PVX

TCV

TSWV

Fusarium oxysporum

Xanthomonas campestris

Blumeria graminis

TMV

P. syringae

Cladosporiumfulvum

LZ-NB-LRR

NB-LRR

CC-NB-LRR

TIR-NB-LRR

LRR-TM

Pathogen-derived Resistance

The concept of pathogen-derived resistance was first propounded by Sanford and Johnston (1985) who suggested using a portion of a pathogen's own genetic material for host defense against the pathogen itself. The underlying rationale was that certain pathogen-derived molecules (RNAs or proteins) may be critical for viral pathogenesis. Non-functional forms of such molecules could act in a dominant negative manner and interfere with virus replication, assembly or movement. Therefore, expression of such dominant negative forms of the pathogen-derived molecule in host cells may confer virus resistance in the expressing host (Sanford and Johnston, 1985).The feasibility of pathogen-derived resistance was first examined by the expression of the Tobacco mosaic virus (TMV) coat protein (CP) gene in tobacco plants (Abel et al., 1986). When challenged with TMV, transgenic plants expressing the TMV CP either did not display symptoms of TMV infection or showed a delay in symptom development. The use of CP expression to confer resistance was quickly verified for several viruses. This type of pathogen-derived resistance is generally referred to as protein-mediated resistance or CP-mediated resistance. In earlier experiments, the prevailing notion was that the virus resistance level is directly related to the CP expression level of transgenic lines.Further investigations by several laboratories, however, led to the surprising finding that that some transgenic lines with high virus resistance levels in fact did not express any viral CP. Moreover, the CP RNA level was very low or even not detectable in these resistant plants. Subsequent work clarified this apparent discrepancy of non-expressing transgenic plants with virus resistance. We now know that these transgenic lines were resistant to virus because the expressed CP mRNA triggered post-transcriptional gene silencing (PTGS) and provided RNA-mediated resistance to virus by the siRNA pathway.

Post-transcriptional gene silencing as mean to achieve virus resistance

One of the major drawbacks of true PDR, i.e. a desired high level of transgenic expression of viral genes, has been overcome by introducing transgenic virus resistance based on ^‘‘co-suppression^’’ or post-transcriptional gene silencing (PTGS). PTGS, also known as RNA silencing refers to related processes of post-transcriptional control of gene expression found in plants and fungi, and more recently in some animal species, and involving suppression of foreign genetic elements such as viruses and transposons through a specific RNA breakdown mechanism. At least in plants, PTGS serves as an adaptive, antiviral defence system. PTGS was first discovered in transgenic petunia plants in which introduced gene copies of the chalcon synthetase gene together with the endogenous gene copy were co-ordinately (co-)suppressed (Van der Krol et al., 1990; Napoli et al., 1990). Next it was proposed by Lindbo et al. (1993) and Dougherty et al. (1994) as mechanism to explain a number of unexpected outcomes of experiments where virus resistance based on PDR was originally aimed. Indeed, it was frequently found in control experiments using untranslatable versions of viral genes (initially included to serve as negative controls) that similar levels

Table 2

Known silencing suppressing plant viruses

Virus

Genus

Suppression

Protein

ACMV (Voinnet et al., 1999)

BSMV (Yelina et al., 2002)

BWYV (Pfeffer et al., 2002)

CMV (Brigneti et al., 1998)

CPMV (Voinnet et al., 1999)

PCV (Dunoyer et al., 2002)

PVX (Voinnet et al., 2000)

PVY (Brigneti et al., 1998)

RHBV (Bucher et al., 2003)

RYMV (Voinnet et al.,

1999)

TBSV (Voinnet et al., 1999)

TSWV (Bucher et al., 2003)

Begomovirus

Hordeivirus

Polerovirus

Cucumovirus

Comovirus

Pecluvirus

Potexvirus

Potyvirus

Tenuivirus

Sobemovirus

Complete

Partial/sys-

temic

Partial

Complete

Partial/sys-

temic

Complete

AC2

P15

P25

HcPro

NS3

P19

NSs

Tombusvirus Complete

Tospovirus Complete

of transgenic resistance could be obtained. Moreover, it was frequently observed that transgenic plant lines, in which only low levels of transgenic viral RNA accumulated, exhibited the highest levels of resistance, while, instead, plant lines with high accumulating levels of transgenic RNA were poor performers. Further studies, in different systems, revealed that indeed in the resistant plant lines the transgenic RNA was rapidly broken down in a sequence-specific manner. Hence, transgenes with no sequence homology to the host^’s genome can trigger RNA silencing and may do so in a very efficient way. To date transgenic resistance against virtually all major plant viruses, indiscriminate of the nature of their genome (RNA or DNA), has been reported. Although originally found in transgenic plants producing foreign or aberrant RNA, evidence has accumulated that PTGS in plants in fact acts as a natural antiviral defence system by surveying for aberrant RNAs such as double-stranded viral replication intermediates. In addition, the mechanism of a natural resistance gene was found to be based on RNA silencing (Covey et al., 1994). A very strong argument pointing toward the natural role of RNA silencing in antiviral defence is the presence of plant viral gene products that, in turn, suppress this silencing. The PTGS defence system acts remarkably efficient and works very broad, i.e. it can be activated in the plant against virtually any virus species. A simplified model to explain how PTGS results in virus resistance and how it can be enhanced by transgene expression is presented in Fig. 1

The model also accommodates data generated by genetic screens of Arabidopsis by which some factors involved in RNA silencing have been identified (Mourrain et al., 2000; Dalmay et al., 2000, 2001). Transgenically expressed ssRNA is first copied into short dsRNA during a surveillance/amplication process in which a host-encoded RNA-dependent RNA polymerase (RdRP) is involved. Such RdRPs have indeed been identified in various plant species (Schiebel et al., 1998), while genetic screens in Arabidopsis confirmed a crucial involvement in PTGS (in case of single copy sense transgenes). Transgenes with inverted repeats may directly produce dsRNA, without the involvement of an RdRP, while this is also true in case of an RNA virus infection where dsRNA is formed as replication inter-mediates. The dsRNA produced in either way form a trigger to recruit further components required for sequence-specific breakdown of the transgenic RNA, i.e. a Dicer-like dsRNAse, while ssRNA targets are broken down by the so-called RISC-like nuclease complexes, which also serve as sequence-specific targeting complexes due to the incorporation of 21~/23 nt short interfering RNAs (siRNAs). Hence, as a product of PTGS typically small, 21~/23 nt long RNA fragments are formed. These small interfering (si)RNAs probably also play a role in the systemic signal which is spread

through the plant. More recently, PTGS has been reported in animals, a.o. in nematodes (C. elegans) and flies (Drosophila), where it has been shown to be involved in genome defence against transposable elements, but where a possible antiviral role still needs to be substantiated (Fire et al., 1998; Kennerdell and Carthew, 1998; Plasterk, 2002).As plant-infecting viruses have to deal with PTGS-based defence mechanisms, it is not at all a surprise that these viruses can encode a counteracting activity, i.e. a ^‘‘suppressor of gene silencing^’’. Table 2 gives an over-view of viruses that have been shown to produce such a suppressor protein. Suppressors of silencing are often identified in silencing reversal assays, using silenced GFP transgenic plants (Brigneti et al., 1998; Voinnet et al., 1999; Dunoyer et al., 2002). One of the best studied suppressors of RNA silencing is the helper component-protease (HCPro) encoded by potyviruses (see also the contribution of V. Vance in this issue). Distinct viruses may specify suppressors which target distinct steps in the silencing reaction, and which may act stronger or weaker on specific parts of the pathway. As already mentioned above, the finding that plant viruses encode such suppressors is consistent with the idea that RNA silencing operates as an innate antiviral defence mechanism, rather than being a mechanism to control host gene expression (for recent reviews, see Voinnet, 2001; Baulcombe, 2002).

REPLICASE-MEDIATED RESISTANCE

Engineering virus resistance by using genes encoding viral RNA-dependent RNA-polymerases (RdRps) was first reported for TMV (Golemboski et al., 1990). A notable inhibition of virus replication at the inoculation site and at the single-cell level in tobacco transformed with a modified RdRp was found. Resistance appeared to be strain-specific, against infection initiated by both TMV virions and RNA. However, although the 54 kDa protein itself was never detected in transgenic tissue, the finding that a mutant encoding only 20% of the protein was ineffective suggested at that time that the protein was indeed responsible for resistance (Carr et al., 1992). In addition, expression of a modified TMV RdRp containing an unintended insertion of a bacterial transposon sequence conferred resistance against TMV and other tobamoviruses (Donson et al., 1993). Differences between the replicase sequences in the transgene and those of the challenging viruses were not compatible with RNA-based mechanisms, favouring a role of the protein itself. However, the picture of replicase-mediated resistance induced solely by the protein is certainly too simple. The full-length 54 kDa RdRp from another tobamovirus, Pepper mild mottle virus appeared dispensable for resistance induction and plants expressing a truncated construct (30% of the protein) were equally resistant. For this, the co-existence of dual protein- and RNA-mediated protection was suggested (Tenllado et al., 1995 and 1996). A later case of TMV replicase-mediated resistance was definitely attributed to RNA-silencing (Marano and Baulcombe, 1998).

More recently, expression of nine distinct overlapping segments covering the full TMV 183 kDa RdRp generated resistance to TMV at low levels, and protein expression was not required. However, a higher protection was conferred by segments covering the polymerase domain of the protein, acting as dominant-negative mutants (Goregaoker et al., 2000). A par conditio model was proposed, in which an initial RNA-based mechanism was responsible for low-level protection. This mechanism would be conferred by any sequence derived from the TMV genome, including the CP ORF. A more active protein-mediated resistance is then thought to intervene, possibly in conjunction with RNA-mediated mechanisms. Replicase genes of other viruses, such as tobra- (MacFarlane and Davies, 1992), potex- (Braun and Hemenway, 1992), poty- (Audy et al., 1994), alfamo- (Brederode et al., 1995), and cucumoviruses (Anderson et al., 1992; Carr et al., 1994; Zaitlin et al., 1994; Hellwald and Palukaitis, 1995; Wintermantel and Zaitlin, 2000), have also been described as resistance sources. For tobraviruses, a 54 kDa portion of the full 201 kDa replicase of Pea early browning virus (PEBV) induced resistance to high doses of PEBV, as well as to Broad bean yellow band virus, but not to other tobraviruses (McFarlane and Davies, 1992). Since constructs with deletions or substitutions were ineffective, resistance was likely protein‑

mediated, but as for TMV-resistant plants, protein has never been detected. Similarly, for potex- and potyviruses, resistance appeared protein-mediated, since again mutants of the 166 kDa protein of Potato virus X (PVX) or the NIb of Potato virus Y (PVY), carrying deletions or mutations in the conserved GDD domain were ineffective (Audy et al., 1994). A reverse situation was described for alfamoviruses, where only plants expressing high doses of the Alfalfa mosaic virus P2 replicase carrying N-terminal deletions or mutations in the GDD motif, were resistant, as opposed to wild-type proteins (Brederode et al., 1995). Resistance to CMV was obtained by engineering sequences from the 2a replicase (Anderson et al., 1992; Carr et al., 1994; Zaitlin et al., 1994). Two different resistance mechanisms were described, one targeting virus replication at the single-cell level (Carr et al., 1994; Hellwald and Palukaitis, 1995), the other limiting systemic (Wintermantel et al., 1997) or cell-to-cell spread (Hellwald and Palukaitis, 1995; Nguyen et al., 1996). Wintermantel and Zaitlin (2000). Later it was demonstrated that the translatability of the 2a replicase, full or C-terminally truncated, was indeed necessary to interfere with CMV infection. Taken all together, it can be concluded that replicase-derived transgenes are a potent source of resistance, but interpretation of the role of (modified) replicase proteins or their transcripts is not always clear. Perhaps true replicase protein-mediated resistance can add to a basal level of RNA-mediated resistance.

REP PROTEIN-MEDIATED RESISTANCE TO SINGLE-STRANDED DNA VIRUSES

Unlike RNA viruses, the genomes of plant single-stranded DNA viruses do not encode polymerases. Instead, their replication requires interaction between a viral replication-associated protein (Rep) and host polymerases. Geminiviral Rep proteins have been widely exploited to generate resistance. The Rep gene of African cassava mosaic virus (ACMV) inhibited virus replication in protoplasts and induced virus resistance in plants, but, although a correlation between transcript level and resistance was reported, protein expression was not analysed (Hong and Stanley, 1996). A protein-mediated resistance was described with a truncated Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep protein (210 aa), that strongly inhibited virus replication in protoplasts and induced resistance when expressed at high levels (Noris et al., 1996; Brunetti et al., 1997). This dominant negative mutant, lacking a conserved NTP-binding domain, acts by inhibiting the expression of the viral Rep protein and by forming dysfunctional complexes with the viral Rep protein (Lucioli et al., 2003). However, the resistance was in some cases unstable due to transgene silencing (Lucioli et al., 2003). A similar construct, having a further deletion at the C-terminus and encoding the first 129 aa of the protein, induced resistance in a strictly sequence-specific manner in the closely related Tomato yellow leaf curl virus (TYLCV) (Antignus et al., 2004) as well as in TYLCSV (Noris and Tavazza, unpublished). Similar strategies with Rep proteins mutated in the ori- or NTP-binding sites were applied to generate plants resistant to other geminiviruses, such as Bean golden mosaic virus (BGMV) (Hanson and Maxwell, 1999) or ACMV (Sangaré et al., 1999).

MOVEMENT-PROTEIN-MEDIATED RESISTANCE

Compared to CP- or replicase-mediated resistance strategies, the expression of dysfunctional or mutant movement proteins (MP) has been reported to confer broader resistance. Plants transgenic for the MP of TMV (p30), lacking three N-terminal amino acids (Lapidot et al., 1993) or a temperature-sensitive version (Malyshenko et al., 1993) showed a delay in both symptom appearance and infection. The dysfunctional MP (dMP) was thought to act as a dominant negative mutant, interfering with local and systemic movement of the challenging virus. Resistance was also effective against taxonomically distant viruses, since a TMV dMP transgene interfered with systemic spread of the tobravirus Tobacco rattle virus, the caulimovirus Peanut chlorotic streak virus and the nepovirus Tobacco ringspot virus, but not the cucumovirus CMV. Little or no effect was observed in the inoculated leaves, indicating that replication and cell-to-cell movement were not impaired (Cooper et al., 1995). Expression of wild-type MP, on the contrary, generally enhanced virus infection.

In contrast to tobamoviruses, which rely on a single protein for their movement, movement of potex- carla-, hordei- and some furoviruses is mediated by three overlapping ORFs composing the triple gene block (TGB). Expression of a 13 kDa protein of the potexvirus White clover mosaic virus disrupted in a highly conserved domain rendered plants resistant to the homologous virus, as well as to other TGB-containing viruses, but not to TMV (Beck et al., 1994). These findings were substantiated by using mutant forms of the 12 kDa protein of the TGB of PVX, which gave resistance to PVX and other TGB viruses, but were inactive against PVY. Both cell-to-cell and systemic virus spread appeared to be blocked, in spite of the relatively low sequence similarity (30-50%) with the PVX TGB transgene (Seppanen et al., 1997).

The possibility to simultaneously obtain engineered resistance to viruses having a single MP or a TGB was pursued by Ares and co-workers (1998) by engineering both the p24 protein of PVX and the p30 protein of TMV. In spite of the lack of similarity between the two proteins, and of the different genome organization of the potex- and tobamoviruses, systemic resistance was indeed obtained in reciprocal challenges. The postulated mechanism relies on the existence of common functional domains shared by the two proteins, and is supported by the possibility to complement movement-defective PVX and TMV with heterologous MPs. These domains are thought to compete for common cellular factors required for movement and interfere with the movement of the challenging virus. Alternatively, a non-specific host defence response may be activated. In this case however, over-expression of MP should lead to a broader range of resistance, which has so far not been described.

RNA-MEDIATED RESISTANCE

RNA -mediated resistance against RNA viruses

As a spin-off of initially unexplained effects observed in protein-mediated resistance approaches, the role of RNA transcripts of viral transgenes made a major contribution to the discovery of an entirely new field in biology involving sequence-specific RNA breakdown. This post-transcriptional gene silencing (PTGS) process, occurring in plants but also in other eukaryotes, is also known as RNA interference (RNAi) or RNA silencing (Voinnet, 2005; Ding and Voinnet, 2007).

As mentioned above, expression levels of transgene-encoded viral proteins often did not correlate with the level of virus resistance. Indeed, plants expressing the lowest or even undetectable levels of protein often displayed the highest resistance. Subsequent expression of untranslatable transgenes formally demonstrated the involvement of transgenic RNA in resistance. However, also in this case, the expression levels of transgenic RNA seemed inversely proportional to the resistance. Lindbo and coworkers (1993) were the first to connect this observation to the previously observed co-suppression phenomenon (Van der Krol et al., 1990; Napoli et al., 1990) involving post-transcriptional down-regulation of endogenous genes by transgenes of identical sequence. The model of Lindbo et al. (1993) is based on the idea that sequence-specific RNA degradation induced by transgenes will be targeting of all RNAs with sequence identity with the transgene RNA. In the case of viral transgenes, this process results in virus resistance. This model has been confirmed and expanded. The basis of sequence-specific recognition was found to be determined by the generation of small interfering RNA (siRNA) molecules derived from the transgene (Hamilton and Baulcombe, 1999). As these siRNA molecules were also observed in wild-type plants infected with viruses and viroids, it was concluded that the RNA-mediated transgenic approach pre-programmed an existing antiviral defence in plants (Baulcombe, 1996). Further exploiting this knowledge led to constructing inverted repeat (IR) transgenes from which long double-stranded RNA precursors of siRNAs were generated. Utilization of such IR transgene constructs yielded a marked increase in the efficiency of this approach. Where single-stranded sense or anti-sense approaches yielded resistance frequencies of in 5-20% of the transgenic plants, IR transgenes that produce dsRNAs proved to yield up to 90% of all transgenic plants resistant to the homologous virus (Waterhouse et al., 1998; Smith et al., 2000; Waterhouse and Helliwell, 2003). It is thought that this is due to the dsRNAs being fed into a later step in the silencing pathway, since the dsRNA itself is a substrate for the RNaseIII-like enzyme Dicer, without requiring the activity of plant-encoded RdRps to produce dsRNAs. Using IRs, it proved possible to produce effective RNA-mediated resistance to a wide range of RNA viruses, even ones for which sense-RNA-mediated silencing was not effective (Chen et al., 2004; Kalantidis et al., 2004; Nomura et al., 2004; Hilly et al., 2005). Thus, the siRNAs generated in transgenic plants charge RNA-induced silencing complexes (RISC) with sequence-specific antiviral recognition prior to infection. Upon inoculation with a limited number of viral RNA molecules these can be rapidly and effectively targeted and degraded, even before virus-encoded RNA silencing-suppressor proteins are produced to interfere. This contrasts normal infections, where the lag in induction of the RNA silencing response provides viruses the time to mount their suppressor-based counter defence. One of the drawbacks of RNA-mediated resistance is that it is ineffective against viruses whose sequence differs from that of the transgene by more than 10% (De Haan et al., 1992). In order to create broader resistance, Bucher et al. (2006) therefore fused 150 nt fragments of viral sequences of four tospoviruses in a single small chimeric IR construct. This strategy resulted in a high frequency of multiply resistant plants. It is envisaged that by extending the transgene construct with additional viral sequences, resistance could be broadened even further. In addition, since this multiple virus resistance was observed in a high proportion of transgenic lines, this approach can also be applied to plant species for which large numbers of transgenic lines are difficult to obtain. The most recent addition to the palette of options was provided by the Chua lab. Virus resistance was produced by modifying plant microRNA (miRNA) cistrons to produce a range of antiviral artificial miRNAs (Niu et al., 2006; Qu et al., 2007). The durability of this novel approach, producing relatively few antiviral small RNAs compared to the long dsRNA approach, needs to be demonstrated (Garcia and Simón-Mateo, 2006)

RNA -mediated resistance against DNA viruses

As mentioned above, plant cells infected with RNA viruses produce virus-specific siRNAs, which are thought to originate from the breakdown of dsRNA replicative forms or from secondary structures of the viral RNA (Hamilton and Baulcombe, 1999; Molnar et al., 2005). Interestingly, plant pararetro- and DNA viruses like caulimoviruses and geminiviruses are also targets of RNA silencing (Al-Khaff et al., 1998; Lucioli et al., 2003; Chellapan et al., 2004a and 2004b). In some cases, this response can lead to recovery of the plants from virus symptoms (Covey et al., 1997; Al-Khaff et al., 1998; Chellapan et al., 2005), suggesting that RNA silencing is a remarkably broad natural defense mechanism that protects plants from viral invasion (Covey et al., 1997; Ratcliff et al., 1997).

Biotechnological approaches expressing sense and anti-sense RNA in transgenic plants have been employed successfully against Tomato golden mosaic virus (TGMV) (Day et al., 1991), TYLCSV (Bendhamane and Gronenborn, 1997) and TYLCV (Yang et al., 2004), confirming the suggestion that RNA silencing can be harnessed for antiviral defence (Lapidot and Friedman, 2002). In attempts to further improve transgenic resistance, Pooggin et al. (2003) obtained recovery from virus infection in a transient assay using IR constructs containing the common region of the begomovirus Vigna mungo yellow mosaic virus (VMYMV). Gafni and colleagues obtained plants resistant to TYLCV by targeting the CP gene with an IR construct (Zrachya et al., 2006). Similarly, Noris (2004) and Ribeiro (2007) with their respective co-workers produced transgenic plants expressing siRNAs from IR constructs against TYLCSV and Tomato chlorotic mottle virus (ToCMoV), respectively. Though these plants often showed significant delays in symptom development, particularly at low inoculums dosage. In sharp contrast to the situation with RNA viruses, completely immune lines were not observed. This may suggest that viral mRNAs are targets of RNA silencing, and that the success of the strategy depends on the relevance of the targeted gene product in the systemic spread of the virus.

GENERATION OF RESISTANCE BY MEANS OF PLANTIBODIES

As an alternative to protein-mediated PDR or pre-activation of intrinsic virus resistance mechanisms such as RNA silencing, biotechnological approaches offer the opportunity to exploit approaches that are entirely novel to plants. One such approach is the expression of antibodies, commonly used in animals to recognize pathogens. Although the immune system linked to these proteins in animals is not present in plants, affinities of selected antibodies can be high enough to disrupt essential functions of a viral protein in plants. Though technically complex, the generation of single-chain variable fragment (scFv) antibodies, the development of the phage display approach, and the generation of synthetic scFv libraries, have greatly improved the applicability of this strategy (Ziegler and Torrance, 2002).

The first successful use of plantibodies described reduced susceptibility to Artichoke mottle crinkle virus by means of an scFv directed against the CP of the virus (Tavladoraki et al., 1993). Subsequently, only little progress was made for many years. It became clear that the main problem for a broad application of this technique was the instability of many scFvs in plant cells. As most viruses replicate in the cytoplasm, it is necessary to direct scFvs into this cell compartment, which presents problems for the correct folding of scFvs. In some cases, it was demonstrated that fusion to the endoplasmic reticulum (ER) retention signal

"KDEL" stabilises them (Schouten et al., 1996; Conrad and Fiedler, 1998). Fecker et al., (1996) demonstrated that scFvs directed against the CP and the non-structural protein p25 of Beet necrotic yellow vein virus could be expressed in Nicotiana benthamiana plants. However, expression was only possible if the scFvs were targeted to the ER, which seems in contrast with their place of function in the cytosol. TMV infection of transgenic tobacco plants expressing scFvs against the intact CP of TMV can be reduced, even with low expression levels of the scFv (Zimmermann et al., 1998), and lead to immunity upon stabilized expression (Bajrovic et al., 2001). Xiao et al. (2000) generated an scFv from an existing broad range monoclonal antibody (MAb) obtained against Johnson grass mosaic virus, which reacts with several potyviruses. Transformed tobacco plants had lower susceptibility to two potyviruses, PVY and Clover yellow vein virus.

A different approach is based on scFvs directed against non-structural proteins that play an important role in virus replication, such as the viral replicase. Using scFvs generated against the RdRp of Tomato bushy stunt virus (TBSV), Boonrod et al. (2005) obtained N. benthamiana plants with high levels of resistance not only to TBSV, but also to other members of the family Tombusviridae: Red clover necrotic mosaic virus, Cucumber necrotic virus and Turnip crinkle virus. Gargouri-Bouzid et al. (2006) demonstrated that high levels of expression of an scFv directed against the NIa of PVY led to complete protection against PVY. Apart from a structural protein associating with the viral RNA, the nucleocapsid proteins of tospoviruses are known to regulate viral transcription/replication and cell-to-cell movement, and thus might be particularly sensitive to inactivation by an scFv. Indeed it was demonstrated that specific scFvs can interrupt virus replication at an early stage of infection (Prins et al., 2005).

For the future it is expected that increasing knowledge of the structure of antibodies will provide the opportunity to improve their stability (Ewert et al., 2004). In addition, new protein scaffolds will be developed that can be used as protein-binding alternatives to antibodies, which may circumvent the shortcomings of scFvs (Binz and Plückthun, 2005).

TRANSGENE-MEDIATED VIROID RESISTANCE

Despite the fact that viroids - small (246-401 nt) single-stranded circular RNA pathogens - do not code for any protein, viroid infection resembles virus infection in many respects (Tabler and Tsagris, 2004; Flores et al., 2005). Upon mechanical inoculation or by vector transmission, viroids enter the plant cell, replicate autonomously, move from cell to cell through the plasmodesmata, become systemic by long-distance movement via the vascular system, and cause disease symptoms. Most of the (>30) known viroid species including Potato spindle tuber viroid (PSTVd; Pospiviroidae) replicate in the nucleus. The four members of the Avsunviroidae family, of which Avocado sunblotch viroid (ASBVd) is the type species, replicate in the chloroplasts. Importantly, PSTVd and ASBVd infection is associated with the activation of the cytoplasmic RNA-mediated plant defence mechanism, which is manifested by the generation of viroid-specific siRNAs of both orientations (Itaya et al., 2001; Papaefthimiou et al., 2001; Martínez de Alba et al., 2002; Markarian et al., 2004). Nonetheless, viroids evade the silencing machinery to maintain infection, even though their mature RNA genomes move from the nucleus or chloroplast into neighbouring cells. Most plant viruses express silencing suppressor proteins that impede cleavage of their genomes. Since viroids do not encode proteins, they cannot adopt this strategy, but must have evolved a still unknown mechanism to combat RNA silencing.

Previous attempts to create viroid resistance were based on antisense RNA, ribozyme and RNase-mediated approaches. The antisense strategy revealed a high degree of variability among different transgenic potato lines, and did not result in immunity against the targeted PSTVd (Matousek et al., 1994). Antisense RNA targeting of the Citrus exocortis viroid (CEVd) also failed to produce fully resistant tomato plants (Atkins et al., 1995). Highly resistant potato plants were generated by the expression of hammerhead ribozymes that were directed against the PSTVd minus-strand RNA, but not against the PSTVd positive-strand RNA (Yang et al., 1997). However, introduction of ribozymes that were functional in potato failed to mediate PSTVd resistance in tomato. The inefficiency of the anti-PSTVd ribozymes in transgenic tomato may be the result of the ability of PSTVd to replicate much faster in tomato than in potato.

So far, one of the most efficient transgenic approaches to achieve viroid resistance is based on the expression of the yeast dsRNA-specific ribonuclease, PAC-1 (Sano et al., 1997; Ishida et al., 2002). In transgenic potato and chrysanthemum plants, PAC-1 production conferred resistance against PSTVd and Chrysanthemum stunt viroid (CSVd), respectively. Moreover, PAC-1 not only degraded the dsRNA structures of different viroids, but also those of numerous viruses (Watanabe et al., 1995). Thus, production of transgenic plants expressing recombinant dsRNA-specific RNases could be a promising approach to generate future crop plants with resistance to multiple plant RNA pathogens. However, it should be noted that some plants expressing PAC-1 (Berthomé et al., 2000) or a bacterial RNase III (Langenberg et al., 1997) displayed abnormal developmental phenotypes, and in the former case, this was shown to be due to induction of PTGS of plant genes.

In plants, RNAi is a major molecular defence mechanism against viruses (this review; Voinnet, 2005; Ding and Voinnet, 2007). Although primarily initiated by dsRNA in the cytoplasm, RNAi is also activated in viroid-infected plants. Double-stranded replicative intermediates of viroids are generated in the nucleus or in chloroplasts (Tabler and Tsagris, 2004) where they can hardly trigger RNAi. It appeared that the rod-like-structured mature Pospiviroidae RNA is processed into siRNAs. Nonetheless, these siRNAs do not prevent viroid infection. The viroid RNA structure, and probably association of the RNA with host proteins, may account for resistance of viroids against RNAi. However, recently, transgenic tomato plants expressing a PSTVd-specific inverted repeat construct (Wang et al., 2004) and accumulating high concentrations of PSTVd-specific siRNAs prior to PSTVd inoculation were found to be PSTVd resistant (Wassenegger, unpublished data). This finding suggests that the RNAi machinery can be charged for increased cleavage of PSTVd and resistance.

As PTGS results in well-fragmented RNA, it might be argued that transgenic resistance against viruses based on this mechanism fulfils more easily the current high demands with respect to biosafety. Indeed PTGS-based resistance does not involve the transgenic production of functional viral genes or proteins, nor does it lead to the presence of transgenic RNA, which might become engaged in RNA recombination events.

The siRNA mediated resistance:

In addition to CP mRNA, RNA-mediated virus resistance can be brought about by expression of satellite RNA, defective interfering (DI) RNA or even noncoding region of viral genome RNAs which compete and interfere with virus replication (Baulcombe, 1996). This type of resistance can also be accomplished by expression of viral sequences in the sense or antisense orientation (Smith et al., 1994; Waterhouse et al., 1998) or in double-stranded forms (Helliwell and Waterhouse, 2003). In all these cases, expression triggers degradation of both the transgene RNA and the corresponding viral RNA via the siRNA pathway.

The siRNA Pathway

The siRNA pathway targets double-stranded (ds) RNA for degradation by DICER-like proteins (DCLs) in a sequence-specific manner through the production of siRNA. Whereas DCL2 cleaves dsRNAs from replicating viruses (Xie et al., 2004), DCL3 cleaves dsRNAs derived from endogenous transcripts through the activity of RDR2 and RDR6 (Dalmay et al., 2001; Mourrain et al., 2000). The siRNAs produced are incorporated into RNA-induced silencing complexes (RISC), which guide cleavage of target RNAs. In RISC, siRNAs mediate sequence-specific binding and cleavage of target RNAs (Baulcombe, 2004). Once cleaved, the RNA is further degraded by exonucleases in the cytoplasm. Alternatively, siRNAs are used as primers for RDR polymerase, using target RNA as a template to generate more dsRNA and produce additional siRNAs. This RDR activity expands the pool of siRNA and amplifies PTGS resulting in more potent silencing activity and effective defense against plant viruses.

Plant MicroRNA (miRNA) Genes and the miRNA Pathway

Recently, novel small RNAs, known as micro-RNAs (miRNAs), have been identified as important regulators of gene expression in both plants and animals. miRNAs are single-stranded RNAs 21 nucleotides (nt) in length, generated from processing of longer pre-miRNA precursors (Bartel, 2004) by DCL1 in Arabidopsis (Xie et al., 2004). These small RNAs are recruited to the RISC complex. Using RNA:RNA base-pairing, miRNAs direct RISC in a sequence-specific manner to down-regulate target mRNAs in one of two ways. Limited miRNA:mRNA base-pairing results in translational repression, which is the case with majority of the animal miRNAs studied so far. By contrast, most plant miRNAs show extensive base-pairing to, and guide cleavage of, their target mRNAs (Jones-Rhoades et al., 2006). So far, 117 miRNA genes have been identified in A. thaliana and Arabidopsis miRNAs have been shown to be important regulators of plant developmental processes (Jones-Rhoades et al., 2006).

The Use of Artificial miRNAs (amiRNAs) to Confer Virus Resistance

Previous reports have shown that alterations of several nucleotides within a miRNA 21-nt sequence do not affect its biogenesis and maturation (Guo et al., 2005; Vaucheret et al., 2004). This finding raises the possibility to modify miRNA sequence to target specific transcripts, originally not under miRNA control. In human cells, miR30 precursor has been modified to generate an amiRNA to down-regulate gene expression probably by translational inhibition (Boden et al., 2004; Dickins et al., 2005; Stegmeier et al., 2005; Zeng et al., 2002). In plants, Schwab et al. (2006) (Schwab et al., 2006) and Alvarez et al (2006) (Alvarez et al., 2006) have recently reported the successful down-regulation of Arabidopsis gene expression by amiRNAs targeting either individual transcripts or groups of endogenous transcripts. These amiRNAs were constructed using precursors of miR164b, miR172a and miR319a as backbones.In an independent study, we have also successfully produced several different amiRNAs using pre-miR159a and pre-miR169c as backbones (Niu et al, 2006). High levels of amiRNAs can be detected in transient expression assays using leaves of Nicotiana benthamiana and also in transgenic Arabidopsis thaliana carrying 35S-amiRNA transgenes. (Fig. 1). The essential features of the application of this technology for virus resistance are outlined in Fig. 2.

To explore possible biotechnological applications of the amiRNA technology, we used pre-miR159a to generate artificial pre-miRNAs¹⁵⁹ (pre-amiRNAs¹⁵⁹) containing sequences complementary to genomes of two plant viruses, Turnip yellow mosaic virus (TYMV) and Turnip mosaic virus (TuMV). Transgenic lines carrying both 35S-pre-amiRNA¹⁵⁹ transgenes can express the appropriate amiRNA at high levels.

Moreover, amiRNA transgenic plants showed specific resistance to either TYMV or TuMV, depending on the expression of the cognate amiRNA (Niu et al., 2006). Finally, transgenic plants that expressed both amiRNAs were resistant to both viruses and the virus resistance trait is heritable through at least 3 generations.

It has been reported that virus and transgene-mediated RNA-silencing becomes attenuated at low temperatures which inhibit siRNA accumulation in insect, plant and mammalian cells (Fortier and Belote, 2000; Kameda et al., 2004; Szittya et al., 2003) This temperature sensitivity explains why siRNA-mediated virus resistance breaks down at 15 degrees C (Szittya et al., 2003). We found that miRNA accumulation was hardly affected at low temperatures. Consistent with this finding, transgenic lines expressing amiRNAs maintained their specific virus resistance even at low temperatures (Niu et al., 2006).

Other strategies (Pokeweed antiviral proteins)

A further strategy for introducing virus resistance in plants is the use of specific and natural inhibitors of virus replication. One promising category of candidate factors is the pokeweed antiviral proteins (PAPs) isolated from several organs of Phytolacca Americana (Pokeweed). PAPs are single-chain ribosome-inactivating proteins (RIBs) and are therefore, upon application in antiviral protocols, potentially also toxic to the host plant (for reviews, see Wang and Tumer, 2000; Tumer et al., 1999; Van Damme et al., 2001; Nielsen and Boston, 2001). Various less toxic types and non-toxic forms have been found or engineered which confer resistance to viruses upon transgenic expression without the detrimental effects of ribosome inactivation (Wang et al., 1998; Zoubenko et al., 2000).

Oligoadenylate synthetase

During the 1990s, it has been tested whether the reconstruction of the mammalian 2?,5?-oligoadenylate

(2-5A) pathway into the plant kingdom would achieve multiple virus resistance. Rather broad virus resistance was indeed obtained by transforming host plants with the mammalian 2-5A synthetase (Truve et al., 1993, 1994), but despite the great potential of this methodology it has not been further exploited since. defence mechanisms of plants towards viruses have been obtained. By exploiting our increased knowledge of the molecular genetics of plant viruses and*/not the least*/the host plant, various antiviral strategies have successfully been developed over the past 15 years or so. Further exploitation of at least part of these strategies, however, seems to have hampered by growing societal concern, especially in Europe, with respect to the production of transgenic food. Therefore, it seems likely that in the near future two approaches will be further applied, i.e. the use of well-defined natural R genes, the functioning of which is expected to be fully unraveled within the next 10 years and which can be introduced very rapidly by marker-assisted breeding techniques, and the further exploitation of another type of natural defence, PTGS.

CONCLUSIONS: EFFICACY, DURABILITY, SAFETY

Considering the number of strategies that have been developed for creating virus and viroid resistance, this area must certainly be considered one of the major success stories in plant biotechnology. However, nearly all of these strategies have never been taken past the stage of proof of principle in the lab or at best small-scale field trials. Indeed it is striking that the only plants that have been grown on a large (i.e. commercial) scale were transformed with complete viral transgenes, and that it appears that the resistance is in all cases of the RNA-mediated type. As a result, our understanding of the field durability of the diverse resistance strategies is truly quite limited. This suggests that the choice of a resistance strategy should take into consideration not only the likelihood of obtaining effective resistance in the greenhouse and small-scale field trials, but also the breadth of sequence diversity of the

target virus, since this would be expected to have an important impact on the probability of resistance breakdown. This also means that it is premature to reject outright resistance strategies based on expression of viral proteins on the grounds that they present a greater potential risk of recombination; in some cases, these may be the best choice in terms of efficacy and durability of resistance, and it is certainly possible to evaluate the potential risks in a given virus/host system.

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Sangaré, A., Deng, D., Fauquet C.M. and Beachy, R.N. (1997) Resistance to African cassava mosaic virus conferred by a mutant of the putative NTP-binding domain of the Rep gene (AC1) in Nicotiana benthamiana. Mol. Biol. Reports, 5, 95–102
Schiebel, W., Pelissier, T., Riedel, L., Thalmeir, S., Schiebel, R., Kempe, D., Lottspeich, F., Sanger, H.L., Wassenegger, M., 1998. Isolation of an RNA-dependent RNA polymerase-specific cDNA clone from tomato. Plant Cell 10, 2087~/2101.

Schillberg, S., Zimmermann, S., Zhang, M.Y., Fischer, R., 2001. Antibody-based resistance to plant pathogens. Transgenic Res. 10

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Liver Cancer

2009-08-25T08:43:00.000-07:00

A Review on Liver cancer:

INTRODUCTION:

Primary liver cancer is the fifth most common cancer world wide and third most cause of death from cancer resulting in more than 600,000 death per year.The major risk factors for hepatocellular carcinoma are chronic hepatitis B or hepatitis c Virus.Alcoholic cirrhosis,and non alcoholic steatohepatitis[1]cancer probably develop in the cirrhotic liver through the induction of accelerated cycles of cell injury,death,and regeneration in an altered fibrotic and inflammatory microenviroenviroment .Abnormal immortalized cell clones arise,and these cells develop genetic and epigenetic alteration.[2]Hepatocellular carcinoma(HCC)is the most common form of liver cancer.This cancer is not only one of the most frequent,but it is also one of the most serious problem,as it usually has a poor prognosis and very lmited means of treatment.[3]It is responsible for 90% of malignant liver tumors in adults.In france,as in other Western countries,the incidence of this cancer has considerably increased over the last 20 years,mainly because of the increased number of cases of cirrhosis caused by hepatitis C virus.Hepatocellular carcinoma develop after cirrhosis.[4]Therefore hepatic function is damaged on these basis live plays a vital role in the production and catabolism plasma lipoproteins.It depends on the integrity of cellular function of liver,which ensures homeostasis of lipid lipoproteins metabolismThere is also some discussion about high density lipoprotein(HDL) metabolism in liver cancer.Cirrhotic patients with hereditary hemochromatosis(HHC)have an increased risk of primary liver cancer(PLC).Primary liver cancer was found in the explants of 10 of 37 patients(27%)and was unsuspected in 7 of 10(70%).[5]The death rate due to HCC has been increased over the last two decadesThe main causes of HCC are the hepatitis B virus(HBV),HCV,aflatoxin B1,alcohol,hemochromatosis,with lower magnitude of risk.

An effective vaccine has been available for prevention of new HbV,however,no vaccine exists against HCV infection

Risk Factors.

The major etiologies of HCC are well defined and include among the well known factors an elevated body mass index, especially in men 5 as well as diabetes mellitus 6 and some of the steps involved in the molecular pathogenesis of HCC have been elucidated in

recent years. As for most type of cancer,hepatocarcinogenesis is a multistep process involving different genetic alterations that ultimately lead to malignant

transformation of the hepatocyte.

HCCs are phenotypically (morphology, microscopy) and

genetically very heterogenous tumors. Induced by chronic liver injury and regeneration in a context of inflammation, immune response and oxidative DNA damage.This may result in genetic alterations, such as the activation of cellular oncogenes, the inactivation of tumor suppressor genes, possibly in cooperation with genomic instability, including DNA mismatch repair defects and impaired chromosomal segregation, overexpression of growth and angiogenic factors, and telomerase activation Chronic viral hepatitis B, C and D, alcohol, metabolic liver diseases such as hemochromatosis and alfa-1-antitrypsin deficiency as well as non-alcoholic fatty liver disease may act predominantly through this pathway of chronic liver injury, regeneration, and cirrhosis. Accordingly, the major clinical risk factor for HCC development is liver cirrhosis since 70-90% of HCCs develop in a cirrhotic liver. [6]

Physiological function of HDL

HDL are a hetrogenous mixture of spherical macomolecules which differ in size(80-120A)and chemical composition[7].ApoA-is present on the majority of HDL particals[8].It is involved in the metabolic intreconvesions that occur as a result of cholesteryl ester transferprotein(CETP),lecithin;cholesterol acyltransferase(LCAT)and lipase activities and their role in the formation of mature HDL.[9]The liver is a major source of the plasma lipoprotein.[10]These facts indicate that the putative primary site of human HDL synthesis is in the liver[11].Epiddemiological studies have shown that obesity is a risk factor for hepatocelular carcinoma[12].

METABOLISM OF HDL IN LIVER

It is relative balance in metabolism of cholesterol and lipoprotein under normal liver function,it was influence of metabolism in lipoprotein with liver disease[13]and an impaired lipid metabolism is often found in patients with chronic liver cancer/diseases.HL activity is suppressed by estradiol and increased by testosterone.In addition ,hepatocellular metabolism of longchain fatty acid suggestthat increased fatty acid oxidation resulting increase in production of hydrogen peroxide,Thus this oxidative stress leads to alternation in gene expressionand in DNA itself.which causes the nonneoplastic liver injury[14]. Liver diseases were classified into chronic hepatitis(CH),liver cirrhosis(LC),hepatocellular carcinoma(HCC),and metastatic liver cancer.Metastatic liver cancer showed a lower HD-fraction level,but higher level of the other parameters than HCC the HDL fraction level in HCC and metastatic liver cancer,and the LDL level in HCC and metastatic liver cancer,differ in survivors and patients who died.Lipid analysis in liver diseases by this method showed results reflecting the pathologic conditions and may be clinically useful.

METABOLISM OF HBV INDUCED HCC

HBV is a partially double stranded hepatotropic DNA virus.HBV infection causes acute or chrinic liver diseases.Recent estimate account about 400 million people chronically infected with HBV.It has been showed that the risk of developing HCC is increased by 100 folds in chronic HBV surface antigen(HBsAG) carriers .HBV infects all age groups,The main problem in patients chronically infected with HBV is the development of progressing chronic liver diseases:chronic hepatitis(CH),cirrhosis and HCC.

COMMON PATHWAY IN HBV AND HCV INDUCED HEPATOCARCINOGENESIS

IT is generally accepted that neither HBV nor HCV are directly cytopathic viruses[15].An important effect of both viruses however ,is causing chronic infection,a repeating attack of the host immune system against the viral infection.Continous cell death,mainly by apoptosis,and reactive regeneration sequence,as the basis of cirrhosis.Chronic inflammation,cell death and proliferation,as a result of oxidative stress,and up and down regulation of several growth factors and cytokines.Viral integration is a essential part of cell transformation by HBV,which does not occur in HCV infection.However,viral proteins,espacialy HBx and to certain extent the core component of HCV,may directly participate in the hepatocarcinogenesis.

Liver Cancer in Atomic-bomb Survivors: Histological

Characteristics and Relationships to Radiation

Histological feature of primary liver cancer among atomic bomb survivors and their relationship to hepatitis B(HBV) and (HCV) infections are of special interest because of the increased risk of liver cancer in persons exposed to ionizing radiation and the high and increasing liver cancer rates in Japan and elsewhere[16]

Follow up data from the life span study(LSS)cohorts of atomic bomb survivors in Japan recently have shown a significant dose response between radiation exposeure and liver cancer,indicating that low linear energy transfer(LET)radiation is capable of causing liver cancer[17]

There are several difficulties in the diagnosis and treatment of this primary liver cancer. Very often, there are no signs of the diseases or the symptoms are non specific (abdominal pain, fever,fatigue,etc.It is only therefore only diagnosed

Late, at an already advanced stage in many cases.Moreover,there are few means of treatment(Surgery,radiotherapy,chemotherapy etc.)

First New Drug Therapy in 30 Years Offers Hope to Liver Cancer Patients

Cancer has approved a new indiction for NEXAVAR(sorafenib tablets)for the treatment of patients with unresectable hepatocellular carcinoma(HCC)or liver cancer.NEXAVAR,an oral anti-cancer treatment,is the first approved drug therapy for liver cancer.

The approval of NEXAVAR in Canada for the treatment of HCC represents a major advance for this patients population.[18]

To find new therapeutic targets

Research is therefore currently being carried out to develop new targeted therapies. From this point of view, the studies of Jean Rosenbaum and his collaborators aim to idetify new mechanisms and new targets in liver cancer. By conducting an analysis of the proteoma1, they compared proteins present in cancer cells with those present in the non-cancerous part of the liver. The researchers then found that certain proteins, and in particular reptin, or RUVBL2, were present in higher quantities in the cancerous part. By collecting a hundred HCC specimens from patients, they also showed that this over-expression of reptin was associated with a worse overall prognosis.

In order to find out if reptin directly plays a role in tumoral progression, they artificially modified its level of expression in human HCC cells. The scientists then found that a reduction in the quantity of reptin in cancer cells stopped cell growth and subsequently caused cell death, whereas an increase in the quantity of reptin made it possible for cancer cells to form larger tumours after their implantation in mice.[19]

while liver resection and transplantation offer the best options for treating hepatocellular

carcinoma (HCC), only 15% of individuals meet the criteria for these surgeries due to the number, size or location of their tumor(s), cirrhosis (and other conditions that cause poor liver function), or other health problems.

Chemoembolization or Transarterial Chemoembolization (TACE)

Chemoembolization involves the insertion of a tiny catheter into the groin, which is threaded up to the hepatic artery. An interventional radiologist injects chemotherapy into the artery, followed by tiny particles to block the flow of blood through the artery. Depending on the type of particles used, the blockage may be temporary or permanent. Although the hepatic artery is blocked,Healthy liver tissue continous to receive blood from hepatic portal vein.[20]

Radiofrequency Ablation (RFA)

Radiofrequency ablation (RFA) involves the use of a probe to deliver thermal energy that destroys cancer cells. This treatment can be performed percutaneously using laparoscopy or by open celiotomy. Numerous non-randomized trials have been published in regards to RFA. Comparison of RFA to other modalities is limited. Reviewed outcomes of 232 patients who were treated with radiofrequency ablation versus those treated by ethanol injection.

Patients had less than three lesions, each less than 3 cm in size. The study endpoints included survival at four years and overall and local disease recurrence.

Results showed a 74% survival with RFA versus 57% survival with ethanol injections.

Summary and perspectives

HCC is one of the most common malignant tumors in some areas of the world with an extremely poor prognosis. HCC treatment is based on randomized controlled trials and many observational studies. Treatment options fall into five main categories: 1) surgical interventions,including tumor resection and LTx; 2) percutaneous interventions, including PEI and RFA; 3) transarterial interventions, including TAC, TAE and TACE, 4) radiation therapy and 5) drugs as well as gene- and immune-therapies. While surgery and percutaneous as well as transarterial interventions are effective in patients with limited disease (up to three lesions <3>80% patients present with multicentric HCC and advanced liver disease or comorbidities that restrict the therapeutic measures to best supportive care. In order to reduce the morbidity and mortality from HCC, therefore, early diagnosis and the development of novel systemic therapies for advanced disease, including drugs, gene- and immune-therapies as well as primary HCC prevention are of paramount importance.

Furthermore, secondary HCC prevention after successful therapeutic interventions needs to be improved to make an impact on the survival of patients with HCC. New technologies, including gene expression profiling and proteomic analyses,[21] should allow to further elucidate the molecular events underlying HCC development and to identify novel diagnostic markers as well as therapeutic and preventive targets.

REFERENCES:

1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-76

2.. Iwakiri Y, Groszmann RJ. The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule. Hepatology 2006;43:Suppl 1:S121-S131.

3. Paris, 30 July 2007 Institute national de la sante et de la recherché medicale

4. 4. Jiang JT, Xu N, Wu CP. Metabolism of high density lipoproteins in liver cancer. World J Gastroenterol 2007;

5. Livw Transplantation and Surgery, Vol 1, No 4 (Jub), 1995: pp 237-241

6..Simonetti RG, Camma C, Fiorello F, et al. (1991). Hepatocellular carcinoma. A worldwide problem and the major risk factors. Dig Dis Sci 36:962–72.

7.Assmann G, Schriewer H. HDL cholesterol: biochemical aspects (author's transl). Klin Wochenschr 1980; 58: 749-756

8. Lewis GF, Rader DJ. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res 2005; 96: 1221-1232

9.Skinner ER. High-density lipoprotein subclasses. Curr Opin Lipidol 1994; 5: 241-247

10.Lewis GF, Rader DJ. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res 2005; 96: 1221-1232

11.Eggerman TL, Hoeg JM, Meng MS, Tombragel A, Bojanovski D, Brewer HB Jr. Differential tissue-specific expression of human apoA-I and apoA-II. J Lipid Res 1991; 32: 821-828

12. Caldwell SH, Crespo DM, Kang HS, Al-Osaimi AM. Obesity and hepatocellular carcinoma. Gastroenterology 2004; 127:S97-S103

13. Garcia A, Barbaras R, Collet X, Bogyo A, Chap H, Perret B. High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2). Biochemistry 1996; 35: 13064-13071

14 Ockner RK, Kaikaus RM, Bass NM. Fatty-acid metabolism and the pathogenesis of hepatocellular carcinoma: review and hypothesis. Hepatology 1993; 18: 669-676

15.. Nakamoto Y, Kanekos S: Mechanisms of viral hepatitis induced liver injury. Curr Mol Med 3: 537-544, 2003

16.1.Department of Pathology, Hiroshima Prefectural Hospital, Hiroshima, Japan; Departments of

2. Epidemiology, 4Radiobiology, and 8Statistics, Radiation Effects Research Foundation,

Hijiyama Park, Minami-ku, Hiroshima 732–0815, Japan. National Academy of Sciences, 2101 Constitution Avenue, Washington

17. Thompson, D. E., Mabuchi, K., Ron, E., Soda, M., Tokunaga, M., Ochikubo, S., Sugimoto, S., Ikeda, T., Terasaki, M., Izumi, S. and Preston, D. L. (1994) Cancer incidence in atomic bomb survivors. Part II: Solid tumors, 1958–1987. Radiat. Res. 137S: 17–67.

18.1 World Health Organization/Hepatitis B. Web site. http://www.who.int/csr/disease/hepatitis/whocdscsrlyo20022/en/. Accessed January 15, 2008.

2. Penn State Milton S. Hershey Medical Center College of Medicine/Malignant Hepatoma. Web site.

http://www.hmc.psu.edu/healthinfo/m/malignanthepatoma.htm. Accessed January 15, 2008.

19. Dartmouth Medical School, Department of Pharmacology and of Genetics, Lebanon, NH 03756 USA.

20.Liver Disease Management and Transplant Program Center for Complex Digestive Disease California Pacific Medical Center P.O. Box 7999 San Francisco, California 94120-7999

(415) 600–1000 www.cpmc.org/liver

Easy to use Protocols for Molecular Biology and Biotechnology Labs

2009-07-15T07:16:00.000-07:00

Primer designing (principles)

1.Determination of particular restriction sites on vector as well as in the primers
2.Make it sure that same restriction site is not present within the coding region of the target gene.
3.Primers should not be complementary to each other in order to avoid primer dimmer formation.
4.Primers should not contain less than 50% GC content
5.Annealing temperature of primers should be considered.

Agarose gel electrophoresis

DNA fragments are separated by electrophoresis on 1% (w/v) agarose gels in 0.5 X TAE buffer containing ethidium bromide (10 mg/ml). Fragment sizes can be estimated by comparining with 1kb DNA ladder.

50X Tris-acetate EDTA buffer (TAE)

Tris base 242 gm

Glacial acetic acid 57.1 ml

0.5 M EDTA (pH 8.0) 100 ml

Make up the final volume with distilled water to 1000 ml.

6X Gel loading buffer

Bromophenol blue 0.25% (w/v)

Xylene cyanol FF 0.25% (w/v)

Glycerol 30.0% (v/v)

Dissolve in distilled water
LB (Luria-Bertini) medium

Tryptone 1.0 %

Yeast extract 0.5 %

NaCl 0.5 %

Agar 1.5 %

Mini prep (Plasmid Isolation) solutions

Solution I (suspension buffer)

Tris (pH 7.4-7.6) 50 mM

EDTA 1 mM

RNase 100 μg/ml

Solution II (Denaturation soln.)

NaOH 0.2 N

SDS 1 %

Solution III (Neutralization soln.)

Potassium acetate 3 M

Glacial acetic acid 11.5ml/100ml (pH 4.8-5.0)
Plasmid isolation from E.coli: alkaline lysis method (miniprep)

Following protocol can be used for the isolation of plasmid DNA from E. coli.

1. Culture a single E. coli colony in 3 ml liquid LB medium containing antibiotics (some times 100 mg/ml ampicillin or 50 μg/ml kanamycin) and grow overnight at 37 °C in an incubator shaker.

2. Centrifuge 1 ml of E. coli culture in 1.5 ml eppendorf tube at 14000 rpm for 1 minute.

3. Discard the supernatant and air dry the pellet.

4. Add 200 ml of solution I to eppendorf tube and suspend the pellet in the solution with the help of vortexer machine.

5. 200 ml of solution II was added to eppendorf tube and mixed well by inverting gently and incubate for 5 minutes at room temperature.

6. Add 200 ml of solution III to eppendorf tube, mix well and incubate for 5 minutes at room temperature then centrifuge at 14000 rpm for 5 minutes.

7. Take the supernatant in a fresh eppendorf tube and add two volume of pre-chilled 100% ethanol or Isopropanol.

8. Incubate the tubes at -20 °C for 20 minutes and then centrifuge at 14000 rpm for 10 minutes.

9. Discard the supernatant, wash the pellet with 70% ethanol and air-dry the pallet.

10. Dissolve the DNA in 20 ml of sterile distilled water was stored at -20 °C.

The plasmid concentration and quality can be detected by 1% agarose gel using stranded DNA markers.
Rapid gel extraction protocol (Quick jet Kit)

NOTE

ü Perform all centrifugations at room temperature.

ü Preheat an aliquot of TE to 65-70 ºC before starting the experiment.

ü Equilibrate a water bath or heat block to 50 ºC.

ü Verify that ethanol has been added to wash buffer (L2).

1. Gel slice excision

Cut the area of gel containing the DNA fragment using a clean, sharp blade. Minimize the amount of surrounding agarose excised with the fragment.

2. Gel slice weighing

Weigh the gel slice.

a. For < 1% agarose gel, place up to 400 mg of gel into a 1.5 ml polypropylene tube.

Divide gel slices exceeding 400 mg among additional tubes. Add 30 μl of Gel solubilization buffer (L1) for every 100 mg of gel.

b. For >1% agarose gel, place up to 400 mg of gel into a 5 ml polypropylene tube.

c. Divide gel slices exceeding 400 mg among additional tubes. Add 60 μl of Gel solubilization buffer (L1) for every 10 mg of gel.

3. Gel solubilization

Incubate at 50 ºC for > 15 min. Mix every 3 min to ensure gel dissolution. After gel slice appear dissolved, incubate for 5 min longer.

4. Cartridge loading

Place a spin cartridge into a 2 ml wash tube. Pipette the mixture from step 3 into the spin cartridge. Centrifuge the mixture in a microcentrifuge at > 12,000 rpm for 1 min. Discard the flow through.

Note: Load no more than 400 mg agarose per cartridge.

5. Optional cartridge wash

Place the spin cartridge back into the 2 ml wash tube. Add 500 μl Gel Solublization Buffer (L1) to the spin cartridge. Incubate at room temperature for 1 min, then centrifuge at > 12,000 rpm for 1 min. Discard the flow through.

6. Cartridge wash

Place the spin cartridge back into the 2 ml wash tube. Add 700 μl of Wash Buffer (L2) (containing ethanol) to the spin cartridge and incubate for 5 min at room temperature. Centrifuge at > 12,000 rpm for 1 min. Discard the flow through. Centrifuge again for 1 minute to remove residual wash buffer.

7. DNA elution

Place the spin cartridge into a 1.5 ml recovery tube (supplied). Add 50 μl of warm TE Buffer (TE) directly to the center of the spin cartridge. Incubate for 1 min at room temperature, then centrifuge at > 12,000 rpm for 2 min.
Phenol-chloroform precipitation of PCR product

Add dH₂O (50 μl) in reaction mixture (50 μl) to make up volume to 100 μl.

Add equal volume phenol : chloroform (100 μl) and mix it.

Centrifuge for 6 minutes

Take supernatant in fresh tube

Add 3M sodium acetate 1/10^th (8 μl pH 5.2) and 2.5 volume absolute ethanol (300 μl).

Incubate at –20 ⁰C for 30 minutes.

Centrifuge for 10 minutes.

Dispose ethanol.

Wash the pallet with 70% ethanol (100 μl), spin for two minutes.

Dry the pallet and dissolve in 15 μl dH₂O.
Preparation of electro-competent cells of E. coli
1. Pick a single colony from a freshly grown plate of E. coli, transfer into 100 ml LB medium in 1 liter flask and overnight incubate at 37 ⁰C with vigorous shaking.
2. Take 2.5 ml of overnight grown culture and transfer to 250 ml LB in 1000 ml flask and shake vigorously at 37 ⁰C until it reach O. D⁶⁰⁰ of 0.5-1.0 (10¹⁰ cells/ml).
3. Then transfer the cells aseptically to sterile disposable 50 ml propylene tube.
4. Keep the culture on ice for 10 minutes.
5. The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in one of volume sterile cold distilled water.
6. Pallet the cells by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspend in 0.5 volume of sterile cold distilled water.
7. Again pellete the cells by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspend in 0.02 volume of sterile cold distilled water.
8. Once again centrifuge at 5000 rpm for 15 minutes and suspended finally in 0.002-0.003 volume sterile 10% cold glycerol.
9. Store the cells in aliquots of 50 μl or 100 μl at -70 ⁰C.
Transformation in E. coli by electroporation
1. Electroporation cuvetts 2 mm gap were placed on ice.
2. Thaw the frozen electrocompetent cells of E.coli on ice.
3. Add 2μl of ligation mixtures into Eppendorf tube containing the competent cells and mixe gently with the pipette tip.
4. The conditions for electroporation are:

Choose mode T 2.5 KV

Set resistance R R5 (129 ohm)

Chamber gap 2 mm

Set charging voltage 2.45 KV

5. Transfer the electrocompetent cells containing the ligation mixture to electroporation cuvette.
6. Give the electiric pulse and add 1 ml of liquid LB medium immediately, mix gently and transfer to a 1.5 ml Eppendorf tube and incubate at 37 ⁰C for 45 minutes with vigorous shaking.
7. Spread 100 μl of transformed culture on solid LB medium having antibiotics.

8. As transformed mixture absorbed completely, seal the plates with sealing film and keep at 37 ⁰C in incubator for over night.

9. Pick single clonies with sterile toothpicks and culture in 3 ml liquid L.B medium containing antibiotic(s).
10. keep culture tubes at 37 ⁰C in water bath for over night with vigorous shaking.

11. Isolate plamid (protocol described above) and verify on 1% agarose gel.

12. Restrict the isolated plasmid DNA suitable restriction enzymes for screening.

13. Mark confirmed colonies, preserve in 50% glycerol and stored at -70 °C for further use.