Transgene Copy Number Research Articles

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Utilization of the Tapestri Platform for Quantification of Single Cell Vector Copy Number in CAR T Cell Products

Chimeric antigen receptor (CAR) T cell immunotherapies have been transformative solutions to treat both adult and pediatric patients with a variety of cancers. Given that CAR T cell therapies involve genetic alterations of a patient's T cells by introduction of the CAR into host cells using lentiviral vectors, the quality of CAR T cell therapies are extensively regulated to warrant safety. Key safety and efficacy attributes need to be accurately measured prior to reintroducing modified T cells back into patients, including CAR transgene copy number or viral vector copy number (VCN). We have previously compared digital PCR (dPCR) strategies to the historical standard release assay of quantitative PCR (qPCR) for VCN quantification given that dPCR is known to provide absolute quantification of target nucleic acid sequences with many advantages over qPCR in regards to simplicity, reproducibility, lower limit of detection, and definitive quantification. We were able to demonstrate that dPCR produces accurate, precise, and reproducible CAR T cell VCN measurements when compared to qPCR. However, these conventional PCR methods measuring vector copy number are either population averaged or involve labor-intensive and time-consuming single cell colony picking and outgrowth, with respective drawbacks in resolution and sample representation. Here, we introduce the Tapestri platform, to measure thousands of single cell level VCN with accuracy and precision. The Tapestri platform workflow is enabled by its two droplet steps. First, thousands of cells are each microfluidically encapsulated in a first droplet where lysis of the cells occur, releasing DNA from its heterochromatin state for uniform DNA interrogation. Then, the individual lysates are uniquely barcoded and target sequences amplified via multiplexed PCR inside each droplet. The final products are sequenced on an Illumina sequencing instrument. Single cell VCN Tapestri data was generated and orthogonally compared with dPCR and matches with the bulk measurement on clonal Jurkat cell lines (VCN=2) mixed with control cell lines (VCN=0) at a range of different ratios with accuracy and precision. We demonstrate that the Tapestri platform provides absolute quantification of CAR transgenes at the single cell level and has the potential to provide more precise measurements of CAR T cell therapeutic product attributes prior to release to patients. Future studies will evaluate the utility of this Tapestri platform in measuring both CAR transgene copies and surface expression/density of the CAR at the single cell level, further improving upon current CAR T cell product release assays.

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

BackgroundGenetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency.ResultsWe have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1 generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes.ConclusionThis new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.

Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs’ low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.

CRISPR/Cas9 - mediated knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

Effects of transgene insertion loci and copy number on Dnmt3L gene silencing through antisense transgene-derived PIWI-interacting RNAs.

PIWI-interacting RNAs (piRNAs), which are germ cell-specific small RNAs, are essential for spermatogenesis. In fetal mouse germ cells, piRNAs are synthesized from sense and antisense RNAs of transposable element sequences for retrotransposon silencing. In a previous study, we reported that transgenic mice expressing antisense-Dnmt3L under the control of the Miwi2 promoter (Tg-Miwi2P-asDnmt3L) exhibited piRNA-mediated DNMT3L down-regulation. In this study, two transgene integration loci (B3 and E1) were identified on chromosome 18 of the Tg-Miwi2P-asDnmt3L mice; these loci were weak piRNA clusters. Crossbreeding was performed to obtain mice with the transgene cassette inserted into a single locus. DNMT3L was silenced and spermatogenesis was severely impaired in mice with the transgene cassette inserted at the B3 locus (Tg-B mice). In contrast, spermatogenesis in mice bearing the transgene at the E1 locus (Tg-E mice) was normal. The number of piRNAs for Dnmt3L in Tg-B mice was eightfold higher than that in Tg-E mice. Therefore, both gene silencing and impaired spermatogenesis depended on the transgene copy number rather than on the insertion loci. Additionally, the endogenous Dnmt3L promoter was not methylated in Tg mice, suggesting that Dnmt3L silencing was caused by post-transcriptional gene silencing. Based on these data, we discuss a piRNA-dependent gene silencing mechanism against novel gene insertions.

Genetic Transformation of Quercus ilex Somatic Embryos with a Gnk2-like Protein That Reveals a Putative Anti-Oomycete Action.

Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm oak plants that overexpress the Ginkbilobin-2 homologous domain gene (Cast_Gnk2-like) that it is known to possess antifungal properties. Proembryogenic masses (PEMs) isolated from four embryogenic lines (Q8, E2, Q10-16 and E00) were used as target explants. PEMs were co-cultured for 5 days with Agrobacterium EHA105pGnk2 and then cultured on selective medium containing kanamycin (kan) and carbenicillin. After 14 weeks on selective medium, the transformation events were observed in somatic embryos of lines Q8 and E2 and a total of 4 transgenic lines were achieved. The presence of the Cast_Gnk2-like gene on transgenic embryos was verified by PCR, and the number of transgene copies and gene expression was estimated by qPCR. Transgenic plants were obtained from all transgenic lines after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In an in vitro tolerance assay with the pathogen P. cinnamomi, we observed that transgenic plants were able to survive longer than wild type.

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR

Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Measurement of Transgenes Copy Number in Wheat Plants Using Droplet Digital PCR.

Genetic transformation is a powerful method for the investigation of gene function and improvement of crop plants. The transgenes copy number in the transgenic line is involved in gene expression level and phenotypes. Additionally, identification of transgene zygosity is important for quantitative assessment of phenotype and for tracking the inheritance of transgenes in progeny generations. Several methods have been developed for estimating the transgene copy number, including southern blot assay and quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization, although convincing and reliable, is a time-consuming method through which the examination of the copy number is challenging in species with large genomes like wheat plants. Although qPCR is potentially simpler to perform, its results lack accuracy and precision, especially to distinguish between one and two copy events in transgenic plants with large genomes. The droplet digital PCR (ddPCR)-based method for investigation of transgenes copy number has been widely used in an array of crops. In this method, the specific primers to amplify target transgenes and reference genes are used as a single duplexed reaction, which is divided into tens of thousands of nanodroplets. The copy number in independent transgenic lines is determined by detection and quantification of droplets using sequence-specific fluorescently labeled probes. This method offers superior accuracy and reliability with a low cost and scalability as other PCR techniques in the investigation of transgenes copy number. This protocol was validated in: Mol Plant (2021), DOI: 10.1016/j.molp.2021.03.022 Graphical abstract Flow chart for the ddPCR protocol.

Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1 (ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1−/−) and observed that human ELP1 expression rescues embryonic development in a dose-dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for nervous system development. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator, their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression.

High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis.

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T1 and T2 progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.

An Afucosylated Anti-CD32b Monoclonal Antibody Induced Platelet-Mediated Adverse Events in a Human Fcγ Receptor Transgenic Mouse Model and Its Potential Human Translatability.

To assess the safety and tolerability of NVS32b, a monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, we used a humanized transgenic (Tg) mouse model that expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs. Prior to its use, we extensively characterized the model. We found expression of all human FCGRs in a pattern similar to humans with some exceptions, such as low CD32 expression on T cells (detected with the pan CD32 antibody but more notably with the CD32b-specific antibody), variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe acute generalized thrombosis in huFCGR mice upon iv dosing. Mechanistic evaluation on huFCGR and human platelets revealed distinct binding, activation, and aggregation driven by NVS32b in both species. In huFCGR mice, the anti-CD32b antibody NVS32b binds platelet CD32a via both Fc and/or complementarity determining region (CDR) causing their activation while in human, NVS32b binding requires platelet preactivation and interaction of platelet CD32a via the Fc portion and an unknown platelet epitope via the CDR portion of NVS32b. We deemed the huFCGR mice to be overpredictive of the NVS32b-associated human thrombotic risk.

Identification of Loci Enabling Stable and High-Level Heterologous Gene Expression.

Efficient and reliable genome engineering technologies have yet to be developed for diatoms. The delivery of DNA in diatoms results in the random integration of multiple copies, quite often leading to heterogeneous gene activity, as well as host instability. Transgenic diatoms are generally selected on the basis of transgene expression or high enzyme activity, without consideration of the copy number or the integration locus. Here, we propose an integrated pipeline for the diatom, Phaeodactylum tricornutum, that accurately quantifies transgene activity using a β-glucuronidase assay and the number of transgene copies integrated into the genome through Droplet Digital PCR (ddPCR). An exhaustive and systematic analysis performed on 93 strains indicated that 42% of them exhibited high β-glucuronidase activity. Though most were attributed to high transgene copy numbers, we succeeded in isolating single-copy clones, as well as sequencing the integration loci. In addition to demonstrating the impact of the genomic integration site on gene activity, this study identifies integration sites for stable transgene expression in Phaeodactylum tricornutum.

Transgenic mice Cre-dependently expressing mutant polymerase-gamma: novel test-system for pharmacological study of mitoprotective drugs

Introduction: PolG-alpha is a nuclear-encoded enzyme which provides replication and repair of mitochondrial DNA. D257A mutation of PolG-alpha leads to change in the N-terminal ”proofreading” domain, which deprives the enzyme of 3′-5′ exonuclease activity, resulting in accumulation of mutations in the mitochondrial genome.
 Materials and methods: Murine zygotes were microinjected with transgene construction carrying mutant murine Polg coding sequence and GFP coding sequence by a loxP-flanked STOP-cassette. Two Cre-activator strains, CMV-Cre (systemic activation) and Tie2-Cre (endothelial activation), were used for activation of the transgene. To confirm the insertion and Cre-dependent activation of the transgene, genotyping and qPCR copy number measurement of mutant Polg were performed, and GFP fluorescence was assessed.
 Results: Two primary transgenic animals were used as the founders for two lines with copy numbers of transgene ~7 and ~5. After systemic activation, the number of the transgene copies decreases to ~1.0 while endothelial specific activation does not affect the number of transgene copies in tail tissue.
 Discussion: A murine model with spatial control of mutant Polgexpression has been developed. To our knowledge, this is the first transgenic model of tissue-specific mitochondrial dysfunction.
 Conclusion: Transgenic mice Cre-dependent expressing mutant polymerase-gamma are a novel test-system for studying mitochondrial biology and efficacy of mitoprotective drugs.

MAP30 transgenic tobacco lines: from silencing to inducing.

DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6days and the concentrations of 3 and 10μM Azacytidine for 10 and 3days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.

An efficient sorghum transformation system using embryogenic calli derived from mature seeds.

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.