RNAi-mediated Reduction of a Major Ruminant Specific Milk Allergen Using a Transgenic Mouse Model

Abstract

Milk from dairy cows is an important human food. It contains the protein betalactoglobulin (BLG) which is not present in human milk and thought to be a major allergen in the milk of cows. RNA interference (RNAi), mediated by sequencespecific micro RNAs (miRNAs), provides a new molecular tool to reduce the levels of undesirable proteins. In this study, I have screened ten miRNAs targeting the BLG mRNA for their potential to knockdown BLG expression using a cell-based in vitro assay. I identified four miRNAs which showed substantial bovine BLG (bBLG) (78-97%) and ovine BLG (oBLG) (33-94%) knockdown activity in vitro. Tandem miRNA constructs with combinations of these four single miRNAs did not result in significantly increased knockdown efficiency compared to the respective single miRNA constructs in the in vitro assay (P>0.05). Because targeting of two different regions has the potential for greater knockdown efficiency in vivo, I chose a tandem construct which achieved in vitro up to 99% bBLG knockdown as compared to 92% and 74% of bBLG knockdown by the single miRNAs. The tandem construct also showed 90% of oBLG knockdown in comparison to 92% and 69% of oBLG knockdown by the single miRNAs in the in vitro assay which made it an ideal candidate for the subsequent evaluation in transgenic (Tg) mice. For the in vivo studies, the cytomegalovirus (CMV) promoter of the selected miRNA constructs was replaced with the promoter of the mouse milk protein gene for whey acid protein (WAP) which directs the expression of the BLG-specific miRNAs to the lactating mammary gland. The Tg mouse lines generated with the tissue-specific tandem construct and the single miRNAs used for the tandem miRNA construct were then crossed with an oBLG or bBLG expressing Tg mouse line to generate double transgenic (miRNA-BLG) mice for assessing BLG knockdown in vivo. Analysis of the milk proteins demonstrated that the tandem miRNA reduced the levels of oBLG and bBLG in milk from the miRNA-BLG mice up to 96% and 46%, respectively. This study validates the mammary gland specific expression of miRNAs as a promising approach to knockdown allergenic proteins in milk. Keywords: BLG, miRNA, RNAi, Transgenic mouse, Gene knockdown.