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[SANET-MG] genetic modification to make unmadable cows
RNA interference technology is new and its stability after genetic
modification is unpredictable. It is probably be unwise to run out and
buy stock in an RNA interference company.
Published online before print March 27, 2006
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0600813103
Agricultural Sciences
Suppression of prion protein in livestock by RNA interference
Michael C. Golding *, Charles R. Long , Michelle A. Carmell *, Gregory
J. Hannon *, and Mark E. Westhusin
*Watson School of Biological Sciences, Cold Spring Harbor Laboratory,
Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, NY
11724; and Department of Veterinary Physiology, College of Veterinary
Medicine, Texas A & M University, College Station, TX 77843
Given the difficulty of applying gene knockout technology to species
other than mice, we decided to explore the utility of RNA interference
(RNAi) in silencing the expression of genes in livestock. Short hairpin
RNAs (shRNAs) were designed and screened for their ability to suppress
the expression of caprine and bovine prion protein (PrP). Lentiviral
vectors were used to deliver a transgene expressing GFP and an shRNA
targeting PrP into goat fibroblasts. These cells were then used for
nuclear transplantation to produce a cloned goat fetus, which was
surgically recovered at 81 days of gestation and compared with an
age-matched control derived by natural mating. All tissues examined in
the cloned fetus expressed GFP, and PCR analysis confirmed the presence
of the transgene encoding the PrP shRNA. Most relevant, Western blot
analysis performed on brain tissues comparing the transgenic fetus with
control demonstrated a significant (>90%) decrease in PrP expression
levels. To confirm that similar methodologies could be applied to the
bovine, recombinant virus was injected into the perivitelline space of
bovine ova. After in vitro fertilization and culture, 76% of the
blastocysts exhibited GFP expression, indicative that they expressed
shRNAs targeting PrP. Our results provide strong evidence that the
approach described here will be useful in producing transgenic livestock
conferring potential disease resistance and provide an effective
strategy for suppressing gene expression in a variety of large-animal
models.--
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