Figure 6. Farrerol promotes the generation of gene-targeted mice with germline transmission.

(A) Schematic diagram of the gene targeting strategy at the Cdx2 locus in mouse embryos. A donor vector containing a promoter-less p2A-mCherry was designed for targeting the Cdx2 locus. The underlined trinucleotide represents the PAM, and the sgRNA targeting site is labeled in brown. The length of both the left and right homologous arm are 800 bp. (B) Representative fluorescence images of gene-edited mouse embryos at the Cdx2 locus at the blastocyst stage. (C–D) Effect of different small molecules treatment on gene knock-in frequency at the Cdx2 locus. Knock-in frequency was indicated by the percentage of mCherry⁺ blastocysts in (C), and was confirmed by PCR genotyping analysis using primers amplifying the flanks of the Cdx2 site in (D). Number above each bar, total blastocysts analyzed. (E) Effect of different knock-in strategies on generation of gene-targeting mice. The microinjected 2 cell stage embryos with or without farrerol treatment was transplanted into the pseudo-pregnant mice. The HMEJ mediated knock-in assay was applied as a control. The founder mice were genotyped for gene-targeting frequency analysis. The germline transmission abilities of founder mice were also validated. (F) Representative fluorescence images of blastocysts from homozygous Cdx2-mCherry mice. (G) Representative fluorescence images of Cdx2 mCherry signals in embryos at the stage of E7.5 and E9.5 after homozygous Cdx2-mCherry mice mating with wild-type mice. *p<0.05, **p<0.01, ****p<0.0001, n.s., not significant, χ²-test.

Figure 6—figure supplement 1. Farrerol promotes precise integration in blastocysts and founder mice.

(A) Analysis of knock-in frequencies at the Cdx2 locus in mouse blastocysts treated with indicated doses of molecules by genotyping analysis. The blastocysts were lysed for genomic DNA extraction, then the 5’ and 3’ junction at the Cdx2 locus were amplified for Sanger sequencing. (B) Experimental scheme for targeted Cdx2-p2A-mCherry knock-in founder mouse. (C) Genotyping analysis of gene-targeting mice. Genomic DNA were prepared from mouse tail tips, then the 5’ and 3’ junction at the Cdx2 locus were amplified. H₂O is set as negative control. For positive control (PC), the gDNA of mouse blastocysts which has been successfully knocked-in at Cdx2 site using the same sgRNA and donor, was used as the template for PCR. (D) Precise in-frame integration was further confirmed by Sanger sequencing in the five gene-targeting mice in farrerol treatment group. HAL stands for the left homologous arm and HAR stands for the right homologous arm. **p<0.01, ****p<0.0001, χ²-test.

Figure 6—figure supplement 2. The effects of small molecule intra-cytoplasmic injection on knock-in efficiency.

(A–D) The knock-in efficiencies at the Actb and Cdx2 locus were analyzed in mouse blastocysts. Knock-in frequency was indicated by the percentage of mCherry⁺ cells at the Actb locus in (A) and at the Cdx2 locus in (B), and was indicated by PCR genotyping analysis using primers amplifying the flanks of Actb site in (C) and Cdx2 site in (D). Number above each bar, total blastocysts analyzed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s., not significant, χ²-test.