Construction of mammary gland specific expression plasmid pIN and its expression in vitro and in vivo

Abstract

The aim of this study was to construct a mammary gland specific expression plasmid pIN and validate its function in expressing goat insulin-like growth factor 1 (IGF-1). The backbone plasmid pBC1 contained goat β-casein 5′ arm and β-casein 3′ arm, to target mammary gland-specific gene expression. First, the igf-1 gene was cloned from liver tissue harvested from a Saanen dairy goat and inserted downstream of the β-casein 5' arm. Then the neo gene was amplified from plasmid pCDNA3.1 and placed downstream of the β-casein 3' arm as a positive selection marker. In order to analyze the bioactivity of plasmid pIN, it was transfected into the Bcap-37 cell line and injected into goat mammary gland. Western-blotting and quantitative polymerase chain reaction (PCR) results confirmed the expression of IGF-1 protein and mRNA in transfected Bcap-37 cells. Further studies (RIA) demonstrated that the expression of IGF-1 protein in transfected group was much higher than that in control group (p < 0.05). In vivo results showed that the expression of IGF-1 in injected group was significantly higher than that in control group. All our results provide evidence that pIN is a mammary gland specific expression plasmids that can target expression of IGF-1 to mammary tissue, with the goal of increasing milk production.

Key words: IGF-1, pIN, Bcap-37 cell line, goat mammary, milk production.