Scientists in New Zealand have combined RNAi interference with nuclear transfer technologies to generate a transgenic cow that produces milk lacking β-lactoglobulin (BLG), the whey protein believed to be the primary cause of milk allergy in people. The team at AgResearch and the University of Waikato say their achievement demonstrates that targeted expression of miRNAs is a viable method for knocking out production of a particular protein in livestock, and will also hopefully allow more research into the “still obscure” function of BLG.
Anower Jabed, Ph.D, Goetz Laible, Ph.D., and colleagues carried out an miRNA screen in BLG-expressing cultured cells to identify molecules that knocked down the protein, and then generated tandem constructs incorporating combinations of two of the most efficient miRNAs that knocked down both ovine and bovine BLG, which are closely related.
The most promising of these tandem miRNA constructs (designated miRNA 6-4) was subsequently tested in a transgenic mouse model that expresses ovine BLG, to confirm its ability to block BLG production in vivo. Having demonstrated activity in the mouse model, the investigators then went on to generate a transgenic calf using nuclear transfer, with a donor cell from a bovine fetal fibroblast cell line transfected with the BLG-silencing miRNA.
Of five resulting pregnancies, one was sacrificed to recover the fetus and obtain transgenic cells for use in future nuclear transfer work. One of the remaining four pregnancies led to birth of a live female cow—albeit without a tail. This calf was hormonally induced to lactate, and analysis of milk samples using SDS-gel electrophoresis, Western blot, and HPLC confirmed that all the samples taken were devoid of any detectable BLG. Interestingly, the lack of BLG had a compensatory effect on the levels of other milk proteins. Compared with natural and induced wild-type milk samples, milk from the transgenic calf exhibited much higher levels of major milk proteins, and in particular the caseins. Of these proteins, α- and β-casein levels were increased more than twofold, and κ-casein levels were elevated about fourfold.
BLG has been suspected as a major milk allergen for decades, but its biological functions and impact on milk composition or nutritional and processing properties remain elusive, primarily because rodents don’t naturally produce the protein, and so unmodified mouse or rat models aren’t available. Reporting on their BLG-deficient calf in PNAS, the researchers say the ability to generate dairy cows with comprehensive knockdown of BLG will enable direct testing of “the longstanding conjecture that milk with greatly reduced BLG level will be less allergenic than normal cows’ milk”. And, they point out, the high casein levels in the BLG-deficient milk should in addition provide for increased calcium levels and high cheese yields.
Moreover, the authors write, the achievement validates miRNA expression as an effective strategy for modifying livestock traits. Drs. Jabed, Laible, and colleagues published their results in a paper titled “Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk.”