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

Background Despite the increasing popularity of deliverable transgenics, a robust and fully validated method for targeting Leydig cells, capable of delivering long‐term transgene expression, is yet to be defined. Objectives We compared three viral vector systems in terms of their cell targeting specificity, longevity of gene expression and impact on targeted cell types when delivered to the interstitial compartment of the mouse testis. Materials & Methods We delivered lentiviral, adenoviral and adeno‐associated (AAV) viral particles to the interstitial compartment of adult mouse testis. Immunolocalization and stereology were performed to characterize ability of vectors to target and deliver transgenes to Leydig cells. Results Viral vectors utilized in this study were found to specifically target Leydig cells when delivered interstitially. Transgene expression in lentiviral‐targeted Leydig cells was detected for 7 days post‐injection before Leydig cells underwent apoptosis. Adenoviral‐delivered transgene expression was detected for 10 days post‐injection with no evidence of targeted cell apoptosis. We found serotype differences in AAV injected testis with AAV serotype 9 targeting a significant proportion of Leydig cells. Targeting efficiency increased to an average of 59.63% (and a maximum of 80%) of Leydig cells with the addition of neuraminidase during injection. In AAV injected testis sections, transgene expression was detectable for up to 50 days post‐injection. Discussion & Conclusion Lentivirus, Adenovirus and Adeno‐Associated virus delivery to the testis resulted in key variances in targeting efficiency of Leydig cells and in longevity of transgene expression, but identified AAV9 + Neuraminidase as an efficient vector system for transgene delivery and long‐term expression. Simple viral delivery procedures and the commercial availability of viral vectors suggests AAV9 + Neuraminidase will be of significant utility to researchers investigating the genetics underpinning Leydig cell function and holds promise to inform the development of novel therapeutics for the treatment of male reproductive disorders.

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Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review

Nakami

Kipyegon

Nguhiu-Mwangi

et al. 2021

Vet World

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Background and Aim: Spermatogonial stem cells (SSCs) have previously been isolated from animals' testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. Materials and Methods: The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. Abstracts were read and relevant full papers were reviewed for data extraction. Results: From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers reported on long-term culture conditions (>1 month) for SSC in different livestock species, 22 papers on short-term cultures (5-15 days), 10 papers on transfection protocols, 18 papers on transplantation using different methods of preparation of livestock recipients, and five papers on donor-derived spermatogenesis. Conclusion: Optimization of SSC long-term culture systems has renewed the possibilities of utilization of these cells in gene-editing technologies to develop transgenic animals. Further, the development of genetically deficient recipients in the endogenous germline layer lends to a future possibility for the utilization of germ cell transplantation in livestock systems.

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

Cited by 4 publications

References 30 publications

Spermatogonial Stem Cells in Farm Animals

Spermatogonial Stem Cells in Farm Animals

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

Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review

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