An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo

Abstract

BackgroundMale germline stem cells (MGSCs) are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.MethodThe present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein (eGFP) transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice (1.5 to 2.0-month-old).ResultsSixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eGFP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of gDNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter, suggesting eGFP transgene was suppressed by DNA methylation in vivo.ConclusionThis research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.Electronic supplementary materialThe online version of this article (doi:10.1186/s40104-015-0058-4) contains supplementary material, which is available to authorized users.

Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

“…The hEF1α promoter also resulted in higher transgene expression in transgenic pigs, which was explained to be due to a hypomethylated status [35]. On the contrary, EGFP transgene expression governed by the CMV promoter, not by the EF-1α promoter, was strongly detectable in offspring generated from gene-modified male mice via lentivirus-mediated male germline stem cell manipulation [36]. Thus, there is a need to confirm which promoter sequence is the most suitable for transgenic dog.…”

A comparison of in vivo viral targeting systems identifies adeno‐associated virus serotype 9 (AAV9) as an effective vector for genetic manipulation of Leydig cells in adult mice

“…Viral vectors have been utilized for the targeting and delivery of transgenes to numerous testicular cell types. The majority of studies utilizing viral vectors to target testicular cells have been focussed on targeting spermatogonial stem cells and their lineage with an overarching aim of rapid production of transgenic lines 18‐24 . This has resulted in varied levels of success 18‐24 .…”

“…The majority of studies utilizing viral vectors to target testicular cells have been focussed on targeting spermatogonial stem cells and their lineage with an overarching aim of rapid production of transgenic lines 18‐24 . This has resulted in varied levels of success 18‐24 . Conversely, methodologies to target Sertoli cells for robust, long‐term delivered transgene expression are now well established with delivery of viral vectors to the Sertoli cells within the seminiferous tubules being almost routine 13,15,25,26 …”

“…In addition to this, dissecting gene function in a mature adult system from its function in a developing tissue can be chal- Gene delivery vectors, in particular viral vectors, have been widely utilized for the delivery of gene manipulation technologies in a number of biological systems including the neurological, 1,2 digestive (liver, small intestine), [3][4][5] ophthalmic, 6 respiratory 7-9 and endocrine (pancreas, male and female reproductive organs) [10][11][12][13][14][15][16][17] with an overarching aim of rapid production of transgenic lines. [18][19][20][21][22][23][24] This has resulted in varied levels of success. [18][19][20][21][22][23][24] Conversely, methodologies to target Sertoli cells for robust, long-term delivered transgene expression are now well established with delivery of viral vectors to the Sertoli cells within the seminiferous tubules being almost routine.…”

Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats

“…Alternatively, direct in vivo germline modification in donor SSCs would obviate the need for recipient animals. In mice, modification of SSCs using viral vectors directly injected into the seminiferous tubule or the interspace of the testes has been reported 131415. However, such direct modification of SSCs has not been performed in other animal models.…”