Lactation Biology 1

Identification of the key genes related to mammary gland development in the dairy goat is important for optimizing milk production. In this research, after comparing the expression of ESTs in 7 Long-SAGE libraries corresponding to different stages of development (early virgin, late virgin, early pregnancy, late pregnancy, middle lactation, early involution and late involution) and sequence alignment with BLAST in the sheep library, we found the gene CAMK2G to be an important gene that affected the development of the mammary gland in virgin dairy goats. mRNA was extracted from healthy virgin mammary glands of dairy goat, and then amplified by RACE (rapid amplification of cDNA ends). We synthesized the 3′and 5′-RACE-fragments by using the CAMK2G’s EST from the virgin mammary gland long-SAGE library as the gene-specific primer, then cloned and sequenced RACE fragments to obtain the full-length gene. The anticipated length of the 3′cDNA of gene CAMK2G was about 500 bp, and 5′cDNA was about 1500 bp. Comparing the homology with the sheep EST library provided by NCBI GenBank confirmed that the cloned gene was CAMK2G. We next performed an RNAi (RNA interference) experiment. The synthesized siRNA was based on the full-length of the gene CAMK2G and transfected into cells that were subcultured from primary epithelial cells of the virgin mammary gland. After the detection of cell viability analyzer and flow cytometry, both the proliferation and the activity of mammary epithelial cells were inhibited (P < 0.05). Real-time PCR showed that the expression of CAMK2G was lower after RNAi (P < 0.01). Addition, Western blotting showed that the expression of β-casein was decreased (P < 0.05). The experiments above were repeated 3 times. Thus, the gene CAMK2G may be important in mammary gland development of the virgin goat.