Research ArticleLIFE SCIENCES

Somatic cell reprogramming-free generation of genetically modified pigs

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Science Advances  14 Sep 2016:
Vol. 2, no. 9, e1600803
DOI: 10.1126/sciadv.1600803
  • Fig. 1 Comparison of the SCNT and the GEEP method.

    In vitro matured pig oocytes are used for SCNT, and in vitro fertilized zygotes are used for GEEP. SCNT involves the removal of oocyte nuclei (enucleation), the transfer of mutant somatic cell nuclei (nuclear transfer), and the activation of the reconstructed embryos after electrofusion, all of which takes ~5 hours. SCNT also requires the generation of mutant donor cells, which takes ~2 weeks. On the other hand, GEEP takes ~10 min to transfer the genome editing system into the zygotes by electroporation. The total manipulation times represent the estimated time required to produce 100 mutant embryos by each method. Mutant embryos are transferred into recipient sows, resulting in the generation of mutant piglets.

  • Fig. 2 Optimization of electroporation conditions for efficient genome editing in pig zygotes.

    (A) Genomic structure of the FGF10 locus and the sgRNA target sequence in the third FGF10 exon. Nucleotides in blue represent the target sequence, and nucleotides in red represent the protospacer adjacent motif (PAM) sequence. (B) Genomic sequences of blastocysts formed after electroporation with Cas9 mRNA and FGF10 sgRNA. Various pulsing conditions were tested (shown on the left) using a fixed voltage (30 V). The arrowhead indicates the Cas9 cleavage sites. (C) Frequency of mutations in the FGF10 target region detected in PCR amplicons. (D) Blastocyst formation rates of the electroporated zygotes. (E to G) Comparison of the genome editing efficiency and blastocyst formation rates between Cas9 mRNA– and Cas9 protein–electroporated zygotes. (E) Direct sequencing of PCR amplicons from the blastocysts after electroporation. (F) Frequency of indels in the FGF10 target region. (G) Blastocyst formation rates of the electroporated zygotes. *P < 0.05 using one-way analysis of variance (ANOVA). Error bars, means ± SEM.

  • Fig. 3 Genome editing of the MSTN gene.

    (A) Genomic structure of the MSTN locus and sgRNA sequences targeting the first MSTN exon. (B) Representative genomic sequences of blastocysts formed after zygote electroporation with Cas9 protein and MSTN sgRNAs (sgRNA1, sgRNA6, and sgRNA7; see also fig. S2). Arrowheads indicate the Cas9 cleavage sites. (C) Frequency of mutations in the PCR amplicons of each target region. (D) Alignment of sequences from each blastocyst after electroporation. Nucleotides in blue indicate target sequences, and nucleotides in red indicate PAM sequences. Nucleotides in green indicate inserted sequences. WT, wild type.

  • Fig. 4 Generation of MSTN mutant piglets.

    (A) Representative target region sequences in MSTN mutant piglets (see also table S1). Nucleotides in blue represent target sequences, and nucleotides in red represent PAM sequences. (B). Expression of MSTN protein in the longissimus thoracis muscle. (C) Phenotypic analysis of the biallelic mutant piglet showed enlarged muscle mass, compared with the WT control (arrowheads). (D) Hematoxylin and eosin staining of the longissimus thoracis muscle. Scale bars, 200 μm. (E and F) Immunohistochemical analysis of fast and slow myosin expression in the muscle. Scale bars, 100 μm. (G) Quantification of slow myofibers in the muscle. (H) Analysis of the genome sequence of F1 blastocysts of piglets #3 and #4. *P < 0.01 using Student’s t test. Error bars, means ± SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1600803/DC1

    Supplementary Text

    fig. S1. Optimization of the time period between IVF initiation and electroporation.

    fig. S2. Genome editing of the MSTN gene.

    table S1. Sequence of the MSTN target region in piglets.

    table S2. Off-target analysis of the piglets by deep sequencing.

    table S3. Sequence analysis of the MSTN gene in piglets #4 and #8 by deep sequencing.

    table S4. Oligonucleotide sequences used to generate sgRNA.

    table S5. Oligonucleotide sequences used for off-target analysis.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • fig. S1. Optimization of the time period between IVF initiation and electroporation.
    • fig. S2. Genome editing of the MSTN gene.
    • table S1. Sequence of the MSTN target region in piglets.
    • table S2. Off-target analysis of the piglets by deep sequencing.
    • table S3. Sequence analysis of the MSTN gene in piglets #4 and #8 by deep sequencing.
    • table S4. Oligonucleotide sequences used to generate sgRNA.
    • table S5. Oligonucleotide sequences used for off-target analysis.

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