Digital PCR (dPCR) and qPCR Mediated Determination of Transgene Copy Number in the Forage Legume White Clover (Trifolium Repens).

Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015–2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito’s siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.

An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.)

Background Despite wheat being a worldwide staple, it is still considered the most difficult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and effective wheat transformation system is still greatly needed. Results We have developed and optimised a reproducible Agrobacterium-mediated transformation system for the spring wheat cv ‘Fielder’ that yields transformation efficiencies of up to 25%. We report on some of the important factors that influence transformation efficiencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for independent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efficiencies might be even higher than those reported. Conclusion We reported here a high-throughput, highly efficient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR–Cas9 based genome editing.

27 OXIDATIVE STRESS OF LIVER IN TRANSGENIC PIGLETS WITH MULTIPLE COPIES OF TRANSGENES SOLUBLE HUMAN TUMOUR NECROSIS FACTOR RECEPTOR TYPE Ig-Fc AND HUMAN HEME OXYGENASE-1

It has been demonstrated that transgene expression is associated with copy number in transgenic animals. Here in, we generated 7 genetically modified pigs expressing both soluble human tumour necrosis factor receptor type Ig-Fc (shTNFRI-Fc) and human heme oxygenase-1 (HO-1). 1 day after Caesarean section, all transgenic cloned piglets showed postnatal death. In the present study, the transgene copy number, H2O2 and superoxide dismutase (SOD) levels in cloned piglet liver were examined to identify the relationship between transgene copy number and oxidative stress of postnatal liver. In this study, 2,209 cloned embryos using somatic cells with 15 copies of shTNFRI-Fc and HO-1 were produced by somatic cell nuclear transfer, and transferred into 6 synchronized recipient sows. Among them, pregnancies were identified in 4 recipients using ultrasonography and only 1 recipient was maintained until full term. In total, 7 cloned piglets were delivered by the Caesarean section. On the next day, they showed postnatal death with clinical symptoms such as dyspnea (Group A). As control group, 292 cloned embryos produced from the cells with at least 4 copies of 2 transgenes shTNFRI-Fc and HO-1 were transferred into a synchronized recipient and pregnancy was identified. Two cloned piglets were delivered normally and maintained healthy. The liver of a live cloned piglet with at least 4 copies (Group B) at 2 days after the Caesarean section was isolated and compared with those of dead 7 cloned piglets (Group A) for HO-1, shTNFRI-Fc, H2O2, and SOD by ELISA analysis. The transgene copy number and expression of shTNFRI-Fc and HO-1 were confirmed by genomic DNA PCR, quantitative real-time PCR (qRT-PCR) and ELISA with appropriate antibodies. Statistical analysis was performed using Graphpad Prism. Level of HO-1, shTNFRI-Fc, H2O2, and SOD ELISA results of each piglets were analysed by unpaired t-test with Welch’s correction. While a transgenic piglet (Group B) had at least 4 copy numbers, all dead cloned piglets (Group A) showed 15 copy numbers. A high level of transgene HO-1 and shTNFRI-Fc expression of liver-derived cells in cloned piglets (Group A) was significantly identified compared with those of a transgenic piglet (Group B) by qRT-PCR and ELISA. While the H2O2 level in cloned piglet liver with 15 copy numbers (Group A) was significantly higher (P < 0.05), the SOD level was lower than those of a cloned pig (Group B; P < 0.05). These results demonstrated that multiple copy numbers could affect the level of oxidative stress in cloned piglet liver. It also affected the transgene expression levels and mortality of cloned piglets. This study was supported by National Research Foundation (#2015R1C1A2A01054373. 2016M3A9B6903410), Ministry of Trade, Industry & Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry and fisheries (#114059–03–2-SB010), Research Institute for Veterinary Science, Natural Balance and the BK21 plus program.