Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis

Abstract

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6–9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives.

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors

“…Injection of retrovirus (Kanatsu-Shinohara et al 2004) or lentivirus (Ikawa et al 2002, Kim et al 2010) into the testis results in infection of multiple cell types including germ cells, Sertoli cells, and/or Leydig cells. Direct testicular injection of a buffered salt solution can be used to introduce shRNA into germ cells, albeit with low transfection efficiency (<5%) (Ho et al 2017). Early gonadal development can be disrupted by cultivating organs in hanging droplets of media supplemented with vivo -morpholinos (Rudigier et al 2017).…”