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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.

SUBMITTER: Ho NR

PROVIDER: S-EPMC5217113 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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

Ho Nicholas R Y NR Usmani Abul R AR Yin Yan Y Ma Liang L Conrad Donald F DF

G3 (Bethesda, Md.) 20170105 1

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 ...[more]

PMID: 27856695

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Project description:BackgroundUsing an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.MethodsUsing the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.ResultsWith first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.ConclusionsExposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.

Dataset Information

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

Publications

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

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