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. 2006 Apr 4;103(14):5285-90.
doi: 10.1073/pnas.0600813103. Epub 2006 Mar 27.

Suppression of prion protein in livestock by RNA interference

Michael C Golding  1 , Charles R Long, Michelle A Carmell, Gregory J Hannon, Mark E Westhusin
Affiliations

Suppression of prion protein in livestock by RNA interference

Michael C Golding et al. Proc Natl Acad Sci U S A. .

Abstract

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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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Design and screening of the lentiviral shRNA expression vector. (A) Graphic representation of the lentiviral shRNA expression system used. This vector is a modification of the plasmid described by Lois et al. (23) with insertion of the mouse H1 RNase P promoter driving expression of an shRNA. The sequence shown here targets the caprine PrP mRNA (accession no. AY723292). (B) Percent suppression of the luciferase-PrP reporter by shRNAs targeting the PrP mRNA sequence. All data are presented as the percent reduction in luciferase activity compared with the control nonrelevant shRNA. Experiments are an average of three independent experiments, and actual percentages and standard deviations for the shRNAs are as follows: E2, 63.7 ± 0.7%; E6, 58.5 ± 0.8%; E7, 2.1 ± 1.2%; E8, 8.2 ± 0.7%; E9, 6.6 ± 1.1%; F2, 48.7 ± 0.8%; F3, 65.9 ± 0.8%; F6, 56.3 ± 0.7%; F9, 80.3 ± 0.7%; and F12, 86.5 ± 0.7%.
Fig. 2.
Fig. 2.
GFP expression in transgenic goat fibroblasts and cloned goat embryos. (A) Fibroblasts are shown during preparation for somatic cell nuclear transfer. (B) Expression of the shRNA-GFP transgene in primary goat fibroblasts after integration of the lentiviral vector. (C) Bright-field image of the hatching goat blastocyst shown in D. (D) Ubiquitin C promoter-driven GFP expression in a hatching blastocyst produced via nuclear transfer by using transgenic goat fibroblasts. Goat embryos were produced by somatic cell nuclear transfer by using GFP-positive transgenic goat cells seen in A and B as nuclear donors. Note the lack of fluorescence in the nondeveloping embryos.
Fig. 3.
Fig. 3.
Expression of green fluorescent protein in whole-mounted tissues from a cloned transgenic fetus (C–H). Images are transmitted (A, C, E, and G) and fluorescent (B–D, F, and H) light micrographs of fresh tissue samples. (A and B) Nontransgenic uterine myometrium from a recipient doe carrying a transgenic fetus. (C and D) Fetal intestinal mesentery. (E and F) Fetal intestinal lumen. (G and H) Fetal liver.
Fig. 4.
Fig. 4.
Characterization of PrP suppression in the transgenic goat. (A) PCR amplification of the shRNA expression cassette from plasmid DNA (control), as well as genomic DNA isolated from WT and transgenic fetal tissue. (B) Western blot analysis of 100 and 75 μg of protein extract taken from WT and transgenic fetal brain. A residual amount of PrP can be detected in the transgenic lane; however, it is substantially reduced when compared with WT. The blot displayed was one of two independent replicates. (C) Immunostaining of placentome cross-sections with an anti-GFP antibody. GFP-positive transgenic cells can be seen surrounding GFP-negative maternal tissue, indicating that expression of the lentivirally delivered GFP is restricted to fetal cells. (D) Negative control placentome.
Fig. 5.
Fig. 5.
Transgenic blastocysts produced by in vitro fertilization and embryo culture. (A) Bright-field image of control blastocyst (noninjected ova). (B) The same embryo as in A viewed using fluorescence microscopy. (C) Bright-field image of a bovine blastocyst that was produced by injection of an in vitro matured bovine ovum with a recombinant lentiviral vector encoding GFP and an shRNA targeting PrP, followed by in vitro fertilization and embryo culture. (D) The same embryo as in C viewed by using fluorescence microscopy. The expression of GFP in the embryo depicted in D demonstrates that this embryo has incorporated the transgene encoding GFP and a shRNA targeting PrP into its genome.
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