Efficient generation of transgenic mice by lentivirus‐mediated modification of spermatozoa

转基因 转基因 生物 绿色荧光蛋白 生殖系 细胞生物学 精子 慢病毒 分子生物学 病毒载体 胚胎 遗传学 生殖技术 基因 胚胎发生 重组DNA 病毒 病毒性疾病
作者
Anil Chandrashekran,Rupa Sarkar,Adrian J. Thrasher,Scott E. Fraser,N. J. Dibb,Colin Casimir,Robert Winston,Carol Readhead
出处
期刊:The FASEB Journal [Wiley]
卷期号:28 (2): 569-576 被引量:15
标识
DOI:10.1096/fj.13-233999
摘要

Transgenic technologies conventionally rely on the oocyte as a substrate for genetic modification. Owing to their accessibility, however, male germ cells, including mature sperm, have material advantages for use in transgenesis. Here we have exploited lentiviruses to generate transgenic animals via the male germline. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally transduced mouse spermatozoa were used in in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥42% of founders were found to be transgenic for GFP. Inverse PCR strategy for integration site analysis demonstrated integration of at least 1 or 2 copies of GFP in the transgenics, mapping to different chromosomes. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals. This relatively simple, yet highly efficient, technique for generating transgenic animals by transducing spermatozoa with lentiviral vectors in vitro is a powerful tool for the study of fertilization/preimplantation development, vertical viral gene transmission, gene function and regulation, and epigenetic inheritance.—Chandrashekran, A., Sarkar, R., Thrasher, A., Fraser, S. E., Dibb, N., Casimir, C., Readhead, C., Winston, R. Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa. FASEB J. 28, 569–576 (2014). www.fasebj.org
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