Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Electroporation in Ascidians: History, Theory and Protocols.

Embryonic development depends on the orchestration of hundreds of regulatory and structural genes to initiate expression at the proper time, in the correct spatial domain(s), and in the amounts required for cells and tissues to become specified, determined, and ultimately to differentiate into a multicellular embryo. One of the key approaches to studying embryonic development is the generation of transgenic animals in which recombinant DNA molecules are transiently or stably introduced into embryos to alter gene expression, to manipulate gene function or to serve as reporters for specific cell types or subcellular compartments. In some model systems, such as the mouse, well-defined approaches for generating transgenic animals have been developed. In other systems, particularly non-model systems, a key challenge is to find a way of introducing molecules or other reagents into cells that produces large numbers of embryos with a minimal effect on normal development. A variety of methods have been developed, including the use of viral vectors, microinjection, and electroporation. Here, I describe how electroporation was adapted to generate transgenic embryos in the ascidian, a nontraditional invertebrate chordate model that is particularly well-suited for studying gene regulatory activity during development. I present a review of the electroporation process, describe how electroporation was first implemented in the ascidian, and provide a series of protocols describing the electroporation process, as implemented in our laboratory.

Production of Transgenic Handmade Cloned Goat (Capra hircus) Embryos by Targeted Integration into Rosa 26 Locus Using Transcription Activator-like Effector Nucleases.

Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC.

Establishment, Growth, Proliferation, and Gene Expression of Buffalo (Bubalus bubalis) Transgenic Fetal Fibroblasts Containing Human Insulin Gene, and Production of Embryos by Handmade Cloning Using These Cells.

The aim of the present study was to compare transgenic cells, containing human insulin gene kept under the control of mammary gland-specific buffalo beta-lactoglobulin promoter, and their counterparts, that is, nontransgenic cells, for examining their potential for the production of embryos following somatic cell nuclear transfer (SCNT). The gene construct was delivered into buffalo fetal fibroblasts (BFF) by nucleofection following which, the transfected cells were selected by culture in the presence of G418 for 3 weeks. Transgene integration into BFF genome was confirmed by polymerase chain reaction (PCR) and reverse transcriptase PCR. At passage 8-10, the growth rate, cell proliferation rate, and quantitative expression of certain genes were compared between transgenic and nontransgenic cells. The growth rate and cell proliferation rate was significantly lower (p < 0.05) for transgenic than for nontransgenic cells. Using quantitative real-time PCR it was found that the expression level of CASPASE 3, CASPASE 9, BAX, and P53 was significantly higher (p < 0.05) and that of HDAC1 and IGF-1R was significantly lower (p < 0.05) in transgenic compared with nontransgenic cells. The differences in the relative expression level of BCL-XL, MCL-1, DNMT1, DNMT3a, GDF9, FGF2, and G6PD between the two groups were not significant. Furthermore, when the two cell types were used as donor cells for production of embryos by handmade cloning, the blastocyst rate was significantly lower (p < 0.05) with transgenic (35.69% ± 1.78%) than with nontransgenic cells (48.75% ± 2.38%). In conclusion, these results indicate that differences were present between transgenic and nontransgenic cells, which may affect the efficiency of SCNT when used as donor cells.