Increased Transformation Efficiency Research Articles

Abstract 619: CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells.

Abstract Reduced DNA repair capacity (DRC) and polymorphisms in genes involved in the repair pathways are associated with increased risk for lung cancer. Studies from our lab have demonstrated an association between increased double strand breaks (DSB) and increased risk for gene promoter hypermethylation. The goal of this study was to identify genes in the DSB repair pathway that may be rate limiting for DNA repair and to determine the effect of gene knockdown on genomic stability, DRC, gene promoter methylation, and cellular transformation. CHK2 is an important DNA damage sensor and cell cycle regulator gene that is important in maintaining genomic stability. DNAPKc is a kinase within the non-homologous end joining (NHEJ) pathway that participates in DNA repair by inducing conformational changes that allows access to DSB sites, and reduced activity is associated with lung cancer. Expression of CHK2 was reduced in 4 of 10 tumor-derived lung cancer cell lines, while 3 cell lines had undetectable levels of DNAPKc. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to carcinogens. CHK2 and DNAPKc were stably knocked down in HBEC2 and HBEC3 to determine their role in DRC, genomic instability and transformation. Knockdown of neither CHK2 nor DNAPKc affected microsatellite instability, nor was deficiency of either gene sufficient to induce spontaneous transformation in HBEC2 and HBEC3. Knockdown of CHK2 or DNAPKc decreased DRC in HBEC2 and HBEC3 in response to bleomycin, (a DSB-inducing carcinogen) indicating an important role for both genes in DSB repair. Knockdown cell lines were treated with bleomycin once a week for 12 weeks to determine the effect of gene deficiency on transformation. DNAPKc deficiency increased transformation efficiency in HBEC2 and HBEC3 by 14- and 4.5- fold, respectively, while CHK2 deficiency increased transformation efficiency in HBEC3 by 6- fold compared to bleomycin treated controls. Studies are ongoing to determine changes induced in the transcriptome and methylome in DNA repair-deficient cells during transformation. These studies will highlight the roles of CHK2 and DNAPKc in DSB repair, malignant transformation, and will shed more light on the role of DNAPKc and CHK2 in reprograming the genome. Supported by R01 ES15262 to SAB Citation Format: Ivo Teneng, Randy Willink, Kieu C. Do, Christopher Dagucon, Steven A. Belinsky. CHK2 and DNAPKc deficiency reduces DNA repair capacity and increases transformation efficiency in human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2013-619

Library construction and evaluation for site saturation mutagenesis

We developed a method for creating and evaluating site-saturation libraries that consistently yields an average of 27.4±3.0 codons of the 32 possible within a pool of 95 transformants. This was verified by sequencing 95 members from 11 independent libraries within the gene encoding alkene reductase OYE 2.6 from Pichia stipitis. Correct PCR primer design as well as a variety of factors that increase transformation efficiency were critical contributors to the method's overall success. We also developed a quantitative analysis of library quality (Q-values) that defines library degeneracy. Q-values can be calculated from standard fluorescence sequencing data (capillary electropherograms) and the degeneracy predicted from an early stage of library construction (pooled plasmids from the initial transformation) closely matched that observed after ca. 1000 library members were sequenced. Based on this experience, we suggest that this analysis can be a useful guide when applying our optimized protocol to new systems, allowing one to focus only on good-quality libraries and reject substandard libraries at an early stage. This advantage is particularly important when lower-throughput screening techniques such as chiral-phase GC must be employed to identify protein variants with desirable properties, e.g., altered stereoselectivities or when multiple codons are targeted for simultaneous randomization.

Agrobacterium-mediated genetic transformation using cotyledons in Japanese pear (Pyrus pyrifolia).

Genetic transformation was successfully established producing both transformed adventitious shoots and calli in Japanese pear (Pyrus pyrifolia Nakai) by using cotyledons as explants. Cotyledons of five cultivars were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying the pBIN19-sgfp, which contained a green fluorescent protein gene and the neomycin phosphotransferase gene. In order to increase transformation efficiency, sonication and ethylenedioxybis (ethylamine)-N,N,N′,N′-tetraacetic acid (EGTA) treatments were applied, which could produce physical wounds across the tissue and prevent plant defense reaction, respectively. Green fluorescent protein (GFP) fluorescence was evaluated two weeks and five months after Agrobacterium inoculation as measures of transient and stable transformations, respectively. As a result, sonication significantly increased both transient and stable expression of GFP fluorescence, whereas EGTA treatment did not show a positive effect on either. Out of 18 regenerated plantlets obtained, one plant regenerated from ‘Agenosho Shinanashi’ showed stable GFP fluorescence. This plant was confirmed as a transformant by PCR and genomic Southern blotting. Three other transformed regenerated shoots by myb gene showed red color, which were derived from ‘Imamuraaki’ by the same transformation method. Transformation system in this study was shown to be reproducible since plural transformants were obtained.

Optimization of Agrobacterium-Mediated Genetic Transformation and Regeneration of Transgenic Plants in Indian Cultivar of Barley (Hordeum vulgare L. cv. BL 2)

An efficient Agrobacterium-mediated genetic transformation protocol has been developed for Indian winter barley. Several parameters affecting Agrobacterium-mediated gene delivery were investigated and optimized. These include use of acetosyringone, surfactants in the infection medium, antinecrotic treatment of explants and heat and centrifugation pre-treatment. Addition of acetosyringone in the infection and co-cultivation media was advantageous and resulted in increase in transformation efficiency from 3.7 to 11.1 %. Addition of surfactants (Tween-20/Pluronic acid F 68) at 0.1 % (v/v) had also resulted in improved gene delivery. Pre-treatment of immature embryos with antinecrotic mixture had significant positive effect on percent callus survival and transformation efficiency. Temperature pre-treatment of immature embryos prior to infection resulted in an increase in the number of GUS positive calli with optimum temperature of 40 °C for 3 min. However, centrifugation of immature embryos had a negative effect on barley transformation. Regenerated transgenic plants obtained after 8 to 10 weeks of selection were further rooted for 2 weeks. The plantlets established in green house condition showed normal growth. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of regenerated transformants, respectively. Molecular analysis of transgenic plants containing II gene (nptII) demonstrated the efficacy of optimised protocol for Agrobacterium-mediated genetic transformation in Indian cultivar of barley for the first time.

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

BackgroundIndustrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies.ResultsWe report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude.ConclusionsE. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

Abstract 1049: DNMT3b increases transformation efficiency and accelerates carcinogen induced transformation of human bronchial epithelial cells

Abstract Malignant tumors and cell lines are characterized by increased expression of DNMT1, DNMT3a and DNMT3b. While evidence suggests that DNMT1, DNMT3a and DNMT3b co-operate in methylation and gene silencing during carcinogenesis, very little is known regarding gene targets for DNMT3a and DNMT3b and their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5mM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and DNMT3b were observed in transformed cells. Increased DNMT3a and DNMT3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and DNMT3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and DNMT3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. Over expression of DNMT3a or DNMT3b did not affect DNA repair capacity and was not sufficient to drive spontaneous transformation as evidenced by inability to form colonies on soft agar. Transformation efficiency was increased 2-2.5 fold in the two HBEC2 cell lines over expressing DNMT3b after 12 weeks of carcinogen treatment relative to treated parental HBEC2. In addition, after 8 weeks, colony formation was evident in the DNMT3b over expressing, but not in parental HBECs indicating that increased expression of this de novo DNMT accelerates transformation. Over expressing DNMT3a had no effect on carcinogen-induced transformation. Current studies are evaluating global re-programming of the epigenome in transformed clones using the Illumina 450k promoter array and the effect of over expression of DNMT3b on stem cell markers. This study was supported by R01 ES008801 to S.A.B. and 1F32 CA157082 to I.T. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1049. doi:1538-7445.AM2012-1049

Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors

Rosellinia necatrix is a fungus that infects a wide range of host plants and ruins a variety of commercially important crops. DNA fragments can be introduced into R. necatrix using conventional protoplast-PEG transformation and genome-integrating vectors; however, transformation efficiency with this strategy is quite low. Therefore, to establish a more effective transformation system for the studies of R. necatrix, an autonomously replicating vector was constructed using AMA1 sequences derived from Aspergillus nidulans, which is distantly related to R. necatrix. Use of this vector with AMA1 sequences increased transformation efficiency in R. necatrix, and the vector was maintained as a plasmid in the transformants. Transient and multivariate functional analyses in R. necatrix were performed using co-transformation of multiple pAMA-H vectors, which each carried either an expression cassette for eGFP, mOrange2, or a geneticin resistance gene. Furthermore, fluorescent proteins expressed from the autonomously replicating vectors were dispersed throughout fungal colonies even though the vectors themselves were restricted to the center of each colony. This intriguing phenomenon indicated that gene products could move from the center to the margin in a colony of the filamentous fungi via a cell-to-cell transport system.

Technological advances in bifidobacterial molecular genetics: application to functional genomics and medical treatments.

Bifidobacteria are well known as beneficial intestinal bacteria that exert health-promoting effects in humans. In addition to physiological and immunological investigations, molecular genetic technologies have been developed and have recently started to be applied to clarify the molecular bases of host-Bifidobacterium interactions. These technologies include transformation technologies and Escherichia coli-Bifidobacterium shuttle vectors that enable heterologous gene expression. In this context, a plasmid artificial modification method that protects the introduced plasmid from the restriction system in host bifidobacteria has recently been developed to increase transformation efficiency. On the other hand, targeted gene inactivation systems, which are vital for functional genomics, seemed far from being practically applicable in bifidobacteria. However, remarkable progress in this technology has recently been achieved, enabling functional genomics in bifidobacteria. Integrated use of these molecular genetic technologies with omics-based analyses will surely boost characterization of the molecular basis underlying beneficial effects of bifidobacteria. Applications of recombinant bifidobacteria to medical treatments have also progressed.

벼 발아초기 종자를 이용한 고효율 단기형질전환 방법

Rice is the most important crop as a model plant for functional genomics of monocotyledons. Rice is usually transformed using Agrobacterium tumefaciens. However, the transformation efficiency using previous method is still low. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the inoculation and co-cultivation step. We directly inoculated Agrobacterium containing a CIPK15 gene under the control of CaMV 35S promoter and NOS terminator in the pCAM1300 vector into the pre-soaked seeds in N6D media for 24 hours. After 7 days of culture at 25℃, calli were formed on seeds cultured on the co-cultivation medium containing an antioxidant compound (1 mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (3 mg/L silver nitrate). We obtained 35 and 22 transgenic plants in rice cultivars, Gopumbyeo and Ilpumbyeo, with increase of transformation efficiency by 30.4% and 22.6%, respectively compared to the general transformation method. The new method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

Agrobacterium-mediated genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] for resistance to legume pod borer Helicoverpa armigera

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L−1 kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L−1 sucrose and 0.5 mg L−1 indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.

High efficiency transformation by electroporation of Yarrowia lipolytica

Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of F. lipolytica, we report here that pretreatment of F. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 10(10)/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform F. lipolytica that will be able to yield an efficiency of 2.1 × 10(4) transformants/ug for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for F. lipolytica transformation.

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures.

BackgroundFollowing genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.ResultsWe identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.ConclusionMost progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

Abstract 2003: Role of de novo DNMTs in carcinogen induced transformation of human bronchial epithelial cells

Abstract Malignant tumors and cell lines are characterized by increased expression of DNMT3a and 3b. While evidence suggests that DNMT1, 3a and 3b co-operate in methylation and gene silencing during carcinogenesis, very little is known about targets for DNMT3a and 3b or their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase /cyclin dependent kinase 4 -immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5µM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC1 and HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and 3b were observed in transformed cells. Increased DNMT3a and 3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and 3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and 3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. HBEC1 and HBEC2 clones that expressed DNMT3a or 3b at levels comparable to those observed in tumor derived cell lines were isolated, expanded and characterized. Over expression of DNMT3a or 3b had no effect on DNA repair capacity, did not affect DNMT1 expression and was not sufficient to drive transformation as evidenced by inability to form colonies on soft agar. Two clones from each cell line over expressing DNMT3a or 3b were treated with carcinogen for one hour, once a week for 12 weeks and analyzed for transformation on soft agar. Studies completed for HBEC1 indicate that expression of DNMT3b increases transformation efficiency. Soft agar analyses for HBEC2 clones are on going. Further studies will characterize the mechanism for increased transformation by examining reprogramming of the epigenome and tumorigenicity of transformed cells in xenografts This study was supported by R01 ES008801. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2011-2003

Improved genetic transformation methods for the model alkaliphile Bacillus halodurans C-125

Bacillus halodurans C-125 is a Gram-positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C-125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. A plasmid artificial modification system (PAM system, Yasui et al. 2008) for B. halodurans C-125 was created that increases transformation efficiency by 10- to 1000-fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. Use of the B. halodurans C-125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C-125. These techniques allow routine genetic manipulation of B. halodurans C-125, a model alkaliphilic bacterium with important industrial properties.

Improved Transformation of Anthurium

Methods to increase transformation efficiency and yields of transgenic Anthurium andraeanum Linden ex. André hybrids were sought while effecting gene transfer for resistance to the two most important pests, bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae) and nematodes (Radopholus similis and Meloidogyne javanica). Differentiated explant tissues, embryogenic calli, and comingled mixtures of the two were transformed with binary DNA plasmid constructs that contained a neomycin phosphotransferase II (nptII) selection gene with a nos promoter and terminator. Explants included ≈1-cm long laminae, petioles, internodes, nodes, and root sections from light- and dark-grown in vitro plants. Bacterial blight resistance genes were NPR1 from Arabidopsis, attacin from Hyalophora cecropia, and T4 lysozyme from the T4 bacteriophage. For nematode resistance, rice cystatin and cowpea trypsin inhibitor genes were used. Cocultivation with Agrobacterium tumefaciens strains EHA105, AGLØ, and LBA4404 ranged from 2 to 14 days. Over 700 independent, putatively transformed lines were selected with 5 and 20 mg·L−1 geneticin (G418) for cultivars Midori and Marian Seefurth, respectively. Putative transgenic lines were selected 1 to 11.5 months, but on average 5.2 to 8.4 months, after cocultivation depending on the tissue type transformed. Significantly more embryogenic calli (one line per 5 mg calli) produced transgenic lines than did explants (one line per 143 mg explants) (P < 0.004) from ≈30 mg of tissue. Calli grew selectively from all explant types, but the type of explant from which each selection was made was not recorded because root, internode, and petiole explants were difficult to discern by the time calli developed. Shoots formed 3 months after calli were transferred to light. Non-transgenic control and transgenic ‘Marian Seefurth’ formed flower buds in the greenhouse ≈28 months after cocultivation. The plants resembled commercially grown plants from a private nursery. No non-transformed escapes were detected among the selections screened for NPTII by enzyme-linked immunosorbent assay and polymerase chain reaction (PCR). The selections were positive for transgenes as assayed by PCR and Southern hybridizations. Southern blots showed single-copy insertions of the NPR1 regulatory gene. The ability to produce large quantities of independent transgenic lines from embryogenic calli in a relatively short time period should enable researchers to evaluate the effectiveness of any transgene by screening numerous anthurium lines for improved performance.

Shuttle Vector System for Methanococcus maripaludis with Improved Transformation Efficiency

We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene

Many researchers have developed various methods for in-planta or floral dip transformation of Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum1, inoculum2, inoculum3 and inoculum4) were used to check the transformation efficiency out of which Inoculum3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7-8 hrs horizontally in low light at 280C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~12-14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines.

An ATM/Chk2-Mediated DNA Damage-Responsive Signaling Pathway Suppresses Epstein-Barr Virus Transformation of Primary Human B Cells

SUMMARYEpstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication nor did the DDR marks co-localize with latent episomes. Rather, a transient period of EBV-induced hyper-proliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoproteins EBNA3C was required to attenuate the EBV-induced DNA damage response We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR which is attenuated by viral latency products to induce cell immortalization.

Rapid recovery- and characterization of transformants following Agrobacterium-mediated T-DNA transfer to sorghum

Although Agrobacterium-mediated transformation of sorghum has been reported, the process is rather lengthy and remains difficult, requiring some very stringent conditions to obtain transformants. We have investigated and describe the parameters related to cocultivation, culture, and regeneration that have allowed us to obtain transgenic sorghum plants in as little as 2.5 months. We observed a 2.9-fold increase in transformation efficiency when L-cysteine was included in the medium during the cocultivation step. Furthermore, the use of modified AB minimal medium, with lower phosphate levels and acidic pH, during the induction of Agrobacterium resulted in a 2.8-fold improvement in transformation efficiencies. Incorporation of an additional binary vector, harboring extra copies of virG and virC genes, in the Agrobacterium did not confer any improvements in the transformation of sorghum. Characterization of transgene activity provided some interesting results suggesting that CaMV 35S promoter activity in T0 generation is very low during the early stages of development of a transgenic sorghum plant, and is not indicative of the expression level during the later stages of development or in the next generation.

Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L.

Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 1 mg l−1 N6-benzyladenine (BA), 30 mg l−1 meropenem and 10 mg l−1 hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5- and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R 2 = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.