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. 2014 Mar;24(3):372-5.
doi: 10.1038/cr.2014.11. Epub 2014 Jan 31.

One-step generation of knockout pigs by zygote injection of CRISPR/Cas system

Tang Hai  1 , Fei Teng  2 , Runfa Guo  1 , Wei Li  1 , Qi Zhou  1
Affiliations

One-step generation of knockout pigs by zygote injection of CRISPR/Cas system

Tang Hai et al. Cell Res. 2014 Mar.
No abstract available

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Figures

Figure 1
Figure 1
Generation of vWF-knockout pigs using the CRISPR/Cas system and phenotypic analyses. (A) Summary of generated vWF mutant pigs via zygote injection of Cas9 mRNA/sgRNA. Two piglets #7 and #8 died soon after birth. Biallelic mutant pigs are #3, #4, #7, #9, #10 and #11. (B) T7 Endonuclease I (T7EI) assay of the pigs produced by zygote injection of Cas9 mRNA/sgRNA from experiment 3. The PCR product encompassing the vWF targeting site of each pig is shown in the upper panel. The denatured and re-annealed PCR product was then digested using T7EI, and the result is shown in the lower panel. The red * indicates the pigs with mutations, the ** indicates the wild-type pig, and the fourth lane shows one biallelic mutant pig that cannot be cut but contains only one type of mutant allele as examined by Sanger sequencing. (C) Sanger sequencing of the targeting site in mutant pigs. The PAM sequence is bold and labeled in red. On the right side of each allele, the sizes of insertions (+) or deletions (Δ) are indicated. (D) Piglets carrying vWF mutation. The red arrowheads mark the biallelic mutant piglets. (E) Western blot detection of vWF expression of monoallelic mutant (Mono), biallelic mutant (Bi) and wild-type (WT) pigs. (F) Plasma vWF antigen levels of Mono, Bi and WT pigs. **P < 0.01, ****P < 0.0001. (G) FVIII activity of Mono, Bi and WT pigs. *P < 0.05. (H) Bleeding time of Mono, Bi and WT pigs. ***P < 0.001. Five wild-type pigs, three monoallelic mutant pigs and five biallelic mutant pigs are included in the assays shown in F-H. The P values are calculated using Student's t-test. Data are presented as mean ± standard deviation (SD).
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