Construction Of Blg Knockdown Cell Lines By Using Lentiviral Vector-Mediated Ran Interference

Goat milk inhibition efficiency of more than 90%is an important milk resources which are affluent in nutrient. However, several major differences of protein component exist between human and goat milk. Beta-lactoglobulin(BLG), which constitute a main part of goat milk, is not easily to be digested and absorbed. It is a major sensitinogen to some infants. To eliminate BLG of milk is an important method to remove the allergy and improve milk quality. Currently, the sensitization of BLG is removed mainly by treating milk with high temperature and pressure, or enzymatic hydrolysis. These methods are complicated and expensive. Our study aims to constructing BLG knockdown cell line by using lentiviral vector-mediated RNA interference, which laid a foundation for the forstering of transgenic goat line with lower BLG content in milk.In this study, we firstly created U6 promoter-driven vectors to express short hairpin RNAs (shRNA) with affinity for different sites on BLG mRNA. Each shRNA expression vector targeted against BLG was co-transfected into the GEF with N1-BLG vector, which was constructed to express goat BLG mRNA in vitro. BLG relative mRNA level in GEF was analyzed by real-time PCR to select the shRNA which inhited BLG mRNA expression most effectively. This shRNA was subsequently transferred into a lentivirus plenti-Lacz vector. The Lentiviral plenti-lacz vector of shRNA targeted against BLG was then transfected into the packaging cells 293T to produce infectious virus. GEF was tranfected with lentivirals followed by Blasticidin selection and culture expansion. PCR sequencing and real-time PCR were used to confirm the incorporation of shRNA expression vector into genome.Our results showed that 3 shRNA expression vectors were constructed targeting against goat BLG gene. One of the three vecotrs can efficiently decrease the expression of BLG with the inhibition efficiency of more than 90%(P<0.01). The vector was recombinated into lentiviral vector to produce infectious virus and then used for GEF transfection. The stable clones were obtained by Blasticidin (4ug/ml) screening after 10-12 d. The stead incorporation of shRNA vector into genome was confirmed by PCR amplification followed by sequencing. Real-time PCR result revealed that the expression of BLG gene was highly inhibited in positive cell clones, the inhibition efficiency was more than 90%. (P<0.01). Our study showed that lentivirus can be used to effectively deliver shRNAs into GEF and cause specific inhibition of BLG expression after transfection.