Scientific Method —

Cow engineered to produce less allergenic milk

Researchers milk mice, make cow that should provide "high cheese yields."

In biology, researchers are usually pretty dismissive of experiments that result in only a single sample. When there's (as scientists put it) an "n of one," it's hard to tell whether the results are significant or a random fluke. But, in the case of a paper released on Monday by PNAS, the n happens to be a cow that carries a transgenic construct that knocks down the expression of a gene that encodes one of the major allergens found in milk. And the data that led to the cow makes a compelling case that the development is significant.

The work focuses on the β-lactoglobulin gene (BLG), which encodes a protein that's found in the milk of cows and other ruminants, but isn't produced by humans. That difference may be what makes it one of the major sources of milk allergies, which affect between two and three percent of infants born every year. Getting rid of the gene might make milk a viable option for more people, provided that doing so has no ill effects on the cows.

But deleting the gene is a real challenge. Unlike mice, where genes can be knocked out routinely, the techniques for elimination of genes and cloning of embryos in the cow are quite a bit less advanced. So, the authors turned to a different approach, called RNA interference. This involves designing short pieces of RNA that match sequences in the messenger RNA produced by the BLG gene, which allows them to base pair and form stretches of double-helical RNA. This keeps the messenger RNA from being translated into the BLG protein.

The authors designed 10 different short RNAs that matched sequences in the BLG gene, then tested pairs of them for their ability to block the production of BLG protein in cultured cells. They managed to find a combination that lowered the amount of BLG made into protein by over 90 percent.

But a cultured cell doesn't actually make any milk. So, the authors turned to a convenient research animal: the mouse. Unfortunately, the mouse (like us humans) doesn't make any BLG protein. So, the authors first had to engineer a construct that caused the mice to produce bovine BLG in their mammary glands. Then they had to insert a second construct, one that produced the interfering RNAs. Then they had to get the mice pregnant and milk them. I am not making this up. Their methods section includes the description, "Milk was collected manually into capillary tubes by gentle massage of teats following oxytocin administration."

Again, it all seemed to work nicely. Mice without the interfering RNAs produced lots of BLG, while those with them barely made any. With the general approach validated, the authors turned to the lengthy and expensive process of making a transgenic cow by injecting the DNA that encodes the interfering RNA into cells in culture, then transferring the nucleus of those cells into a cow's egg in order to make a clone. This process is generally inefficient in many mammals, and often produces defective embryos. Only five pregnancies resulted from 57 cloning attempts, and only one produced a live birth. "Unexpectedly, the miRNA 6–4 calf was born without a tail." Oops.

The authors can't tell whether this was a cloning defect, a defect caused by the insertion of the genes for the interfering RNA, or simply a random genetic defect that has nothing to do with the experiments. Breeding should help sort that out.

Fortunately, the calf was a female, and hormone treatment got it to make milk. Which (no doubt much to the authors' relief), did not contain BLG. In fact, the protein levels in the milk remained constant, as other proteins were increased to compensate for the loss of BLG. These include the caseins, which the authors suggest "should provide for increased calcium levels and high cheese yields."

Given the amount of work involved in developing RNA interference for cows, it's somewhat ironic that the same issue of PNAS contains a report on efficient gene knockouts in livestock. Still, that work was done in pigs, so there's no guarantee it will translate smoothly to cattle. And, in the mean time, the New Zealanders behind the transgenic work are undoubtedly attempting to create an entire allergen-free herd.

PNAS, 2012. DOI: 10.1073/pnas.1210057109  (About DOIs).

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