Sex-Specific Gene Knockdown in Mouse Fetal Germ Cells.

Abstract

Fetal germ cells are the precursors of oocytes and sperm. Gene function during germ cell sex differentiation is often studied through the production and analysis of transgenic and knockout models. However, these techniques are time and resource consuming. We have established a gene knockdown system for mouse fetal germ cells, combining electroporation (EP) and a germ cell culture method. GFP-positive male germ cells were isolated from 13.5 days post coitum (dpc) Oct4-GFP Tg gonads and transfected with shRNA vectors via EP. After EP, the germ cells were placed on mesh inserts and cultured for 2, 4 and 6 days. First, we examined knockdown of Gapdh, a housekeeping gene in male germ cells. Vectors with Gapdh shRNA sequences were transfected into the isolated male germ cells (1.5 –2 × 105 cells). Two days after EP, Gapdh expression in the transfected cells was successfully suppressed to 20% of the non-targeting controls. Next, we attempted knockdown of DNA methyltransferase-like (Dnmt3l). Dnmt3l is essential for establishing male-specific methylation imprints and its expression is initiated in the male germ cells by 15.5 dpc. Dnmt3l shRNA vectors were transfected into the male germ cells. Two days after EP, Dnmt3l expression in the transfected cells was drastically decreased to 10% of the non-targeting control. This level of suppression was stably maintained for 6 days in cultured germ cells. We next performed immunocytochemical staining for DNMT3L. Six days after EP, DNMT3L protein was clearly localized in the nuclei in the control cells. On the contrary, DNMT3L expression was not detected in the germ cells transfected with the Dnmt3l shRNA vector. These results demonstrate that our gene knockdown system should be an effective tool for functional analysis of sex-specific genes in fetal germ cells in vitro. This research was supported by the National Institutes of Health Grant P20RR024206 to YY, National Institutes of Health Grant G12RR003061 to YY, and NIH/NIDDK STEP-UP to SK.