206 Overexpression of germ cell genes DAZL, STRA8, and BOULE in bovine adipose tissue-derived mesenchymal stem cells for male germ cell derivation

In vitro gamete derivation from stem cells has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSC) may be suitable candidates for in vitro gamete derivation given their differentiation capacity and their potential for cell therapy. DAZL (deleted in azoospermia-like) and BOULE (also called BOLL) encode RNA-binding proteins that control differentiation of germ cells; STRA8 (stimulated by RA-8) encodes a protein required for meiosis. Considering the crucial roles of these factors, the aim of the present study was to evaluate co-overexpression of different combinations of DAZL, STRA8, and BOULE on germ cell gene expression profile in adipose tissue-derived MSC (AT-MSC). AT-MSC were harvested from abattoir-derived male bovine fetuses (n = 9; 7-8 months of gestation). The optimal concentration of Lipofectamine 2000 (1, 1.5, 2, 1.5 ng µL−1; Invitrogen) was analysed in AT-MSC transfected with plasmid pSIN-EF2-Puro containing DAZL, STRA8, and BOULE coding sequences (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro). Then, AT-MSC were transfected either with plasmids containing DAZL, STRA8, and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro), DAZL and BOULE genes (pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro), DAZL (pSIN-EF2-DAZL-P2A-T2A-Puro), or empty plasmid at a concentration of 1 ng µL−1 of Lipofectamine. Cell samples were obtained from each plasmid treatment and analysed for expression of housekeeping genes ACTB and GAPDH and germ cell genes DAZL, PIWIl2, STRA8, and BOULE by quantitative-PCR using relative values (Quantity) through the ΔΔCT formula. AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro using 1 ng µL−1 of Lipofectamine achieved higher (P < 0.05) expression of DAZL (13.3-fold) compared with cells transfected with empty vector. Moreover, AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-BOULE-Puro had higher (P < 0.05) levels of DAZL mRNA (3.8-fold) compared with empty vector. Messenger RNA levels of STRA8 (1.4-fold) were only detected in AT-MSC transfected with pSIN-EF2-DAZL-P2A-STRA8-T2A-Puro; PIWIl2 and BOULE were not detected in transfected or untransfected AT-MSC. In conclusion, bovine fetal AT-MSC are amenable for overexpression of germ cell markers DAZL and STRA8. Transfection with plasmid containing three germ cell genes (DAZL, STRA8, and BOULE) allowed overexpression of DAZL, whereas transfection with plasmid containing two germ cell genes (DAZL and STRA8) achieved overexpression of STRA8. This study was supported by Fondecyt grant 1191114, Government of Chile.