Fig. 2
From: Versatile generation of precise gene edits in bovines using SEGCPN
Generation of blastocysts and fetuses with MSTN point mutations using SEGCPN. a Procedure for generating blastocysts and fetuses with MSTN point mutations using SEGCPN. b Identification of MSTN point mutation-edited cell clones by PCR. M, 1-kb DNA ladder; C1–C12, MSTN-edited clones; P, donor vector; WT, wild-type cells; H2O, negative control. c Identification of MSTN point mutation-edited blastocysts by PCR. M, 100-bp DNA ladder; embryo+/−, heterozygous MSTN-edited embryos; embryo−/−, homozygous MSTN-edited embryos; WT, wild-type embryos; H2O, negative control. d Sanger sequencing chromatograms of DNA from WT and MSTN mutant blastocysts. The red arrow indicates the substituted nucleotide. Relevant codon identities at the target site are presented beneath the DNA sequence. e Alignments of mutant sequences from the TA-cloning sequencing of a single embryo. The G.C-to-A.T point mutation site is shown in red. The column on the right indicates the frequencies of mutant alleles. WT, wild-type embryos. f Identification of mosaicism in a heterozygous MSTN point mutation-edited fetus. P4/P5, identification of MSTN+/− gene editing in various fetal tissues. Neo-F/R, the marker gene in the ESSEE detected in various fetal tissues. Cell+/−, heterozygous positive gene-edited clone; lanes 3–9, various tissues of an MSTN+/−-edited fetus; WT, wild-type tissue; P, donor vector; H2O, negative control