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Baylor Team Publishes New Method Of Creating Transgenic RNAi Animals

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Although transgenic RNAi mice have allowed researchers to circumvent many of the limitations of traditional knockout mice, creating the animals is still a time-consuming and expensive process.

However, researchers at Baylor College of Medicine published this week data showing that shRNA-expressing constructs, delivered via conventional pronuclear injection, may be used to create transgenic mice in a quick and relatively cost-effective manner.

"Transgenic RNAi mice have been produced in two ways," according to the paper, which appears online this week in Proceedings of the National Academy of Sciences. "One way has been to first generate mouse [embryonic stem] cell clones that show RNAi effect and then to use the … clones to produce mice. The other is to infect or inject one-cell mouse embryos with lentiviruses carrying an shRNA-expressing cassette."

"Both procedures are pretty labor-intensive and require a lot of expertise," Pumin Zhang, one of the PNAS paper's authors, told RNAi News this week. For the first, "you need ES cells. The other one you have to generate high-titer lentiviruses … and put them into a mouse embryo, which is not a trivial thing."

Zhang said that in looking to overcome these hurdles, he and his colleagues decided to try to create transgenic RNAi mice in the same fashion as one would create any other transgenic mouse.

To test their idea, they made transgenic RNAi vectors expressing shRNA constructs targeting genes associated with kidney development. The vectors were then introduced into mice embryos as transgenes by way of pronuclear injection.


"No transgenic lines need to be established, which eliminates a significant amount of animal costs."

The researchers noted in the PNAS paper that the knockdown effect they achieved "can be transmitted for many generations in [the] transgenic animals. In a small-scale screening for developmental defects in the kidney, we uncovered a potential role of Id4 in the formation of the renal medulla."

The researchers further state that "the effect of knocking down gene expression on development can be scored directly in the embryos injected with a construct. In essence, the mouse embryos are used in a way analogous to the cultured cells for phenotypic observations. No transgenic lines need to be established, which eliminates a significant amount of animal costs," they added.

Zhang noted that the technique he and his colleagues developed would still be rather expensive if used for large-scale screening, but compared with making conventional knockouts, it "is a bargain … [and] right now there is no alternative."

The technique can be modified, as well, he said. "The promoter is just an H1 promoter, so you can adapt inducible promoters or Cre-LoxP systems, and generate tissue-specific or cell-type specific knockdown" without turning to lentiviruses.

— Doug Macron ([email protected])

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