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Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein.

ABSTRACT: Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis.

SUBMITTER: Shinohara T

PROVIDER: S-EPMC7066332 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.ResultsFunctional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.ConclusionsTogether, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.

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Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein.

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