We hypothesized that insulin-induced gene 1 (INSIG1) affects milk fat synthesis in buffalo. For this reason, the protein abundance of INSIG1 in the mammary tissue of buffalo during the peak period of lactation and dry-off period was evaluated. The results showed that the expression of INSIG1 at the peak of lactation was lower than that in the dry-off period. To explore the role of INSIG1 in milk fat synthesis, the buffalo mammary epithelial cells (BMECs) were isolated and purified from buffalo mammary tissue, and INSIG1 gene were overexpressed and knocked down by constructing the recombinant lentivirus vector of INSIG1 gene and transfecting into BMECs. Results revealed that INSIG1 overexpression decreased the expression of INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6, which led to reduction of triglycerides (TAG) content in the cell. In contrast, knockdown of INSIG1 had a positive effect on mRNA expression of the above genes. Overall, the data provide strong support for a key role of INSIG1 in the regulation of milk fat synthesis in BMECs.

Lentiviruses as gene transfer vectors have been used successfully to transfect mammal embryonic stem cells and germline stem cells, but this has not been attempted in avian primordial germ cells (PGCs). PGCs were isolated from the gonads of Isa-brown chicken embryos at stage 28 and co-cultured with gonadal stroma cells. A lentiviral vector pLenti-CMV-EGFP was constructed and the virus harvested by cotransfecting 293FT cells with the vector and packaging plasmids. Concentrated lentiviruses were used to transfect chicken PGCs, the transfection efficiency was up to 24.19%.