Lentiviral Mediated Transgenesis by In Vivo Manipulation of Spermatogonial Stem Cells

Lalit Sehgal,Nileema Khapare,Sorab N Dalal,Rahul Thorat,Mugdha Sawant,Amitabha Mukhopadhaya,David S Milstone

doi:10.1371/journal.pone.0021975

Abstract

This report describes a technique for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells with a high rate of success. Spermatogonial stem cells (SSCs) in pre-pubescent animals were infected in vivo with recombinant lentiviruses expressing EGFP-f and mated with normal females. All male pre-founder mice produced transgenic pups with an overall success rate of over 60%. The transgene was heritable and the pre-founder mice could be used in multiple mating experiments. This technology could be used to perform overexpression/knockdown screens in vivo using bar-coded lentiviruses, thus permitting the design of genetic screens in the mouse. Further, this technology could be adapted to other laboratory animals resulting in the generation of model systems that closely approximate human development and disease.

Highlights

The generation of genetically modified mice has spurred great advances in our understanding of various aspects of growth and development
Recombinant lentiviruses expressing EGFP-f (EGFP tagged with a farnesylation signal) were injected into the intertubular spaces of the testis targeting undifferentiated spermatogonia present in the seminiferous tubules
Transgenic pups were generated from mating experiments with three independently derived pre-founder mice at an overall rate greater than 60% (Figure 1C–E, Table 1), a rate that is much higher than previously reported with conventional transgenic protocols or with retroviral infection of spermatogonial stem cells in vitro or in vivo [6,7]

Summary

Introduction

The generation of genetically modified mice has spurred great advances in our understanding of various aspects of growth and development. Multiple technologies have used either injection into a one celled embryo followed by implantation into a pseudo pregnant mother [1], or used stem cell aggregation techniques to generate either knockout [2] or knockdown mice [3]. These experiments are expensive, labor-intensive, time-consuming and require several female donors. Other methods employed in the recent past have infected fertilized eggs in vitro with recombinant lentiviruses, followed by implantation of the embryo into pseudopregnant females [9] While these methods provided better success rates, the implantation experiments are technically cumbersome and require several female donors. The report did not mention the rate of transgenesis in the pups [11]

Methods

Results

Conclusion