Table 1.
Targeting efficiencies and homozygosity for FLPo knock-in into the DBH locus using CRISPR/Cas9 methods for ES cell electroporation.
Shown are targeting efficiencies based on the locus and ratios of Cas9 and sgRNA expression vector: targeting vector used.
Fig 1.
Generation of the DBH-FLPo mouse line.
A) Targeting schematic. The targeting vector contains a 1kb 5’ homology arm, FLPo recombinase, Lox2722-flanked neomycin cassette, and a 1kb 3’ homology arm. The FLPo recombinase sequence and neomycin cassette was knocked into the start codon of the DBH gene, replacing 106 bps of the first exon. B) PCR genotyping of neomycin selected ES cell clones. Targeted knock-in was determined using PCR primers that spanned from within FLPo to outside each homology arm. Amplification of a band indicates targeting. Shown are the results from a 10:1 px330_DBH_sgRNA1:targeting vector molar ratio.
Fig 2.
Generation of the DBH-p2a-FLPo mouse line.
A) Targeting schematic. The targeting vector contains a 1kb 5’ homology arm, p2a-FLPo recombinase, Lox2722-flanked neomycin cassette, and a 1kb 3’ homology arm. The p2a-FLPo recombinase sequence and neomycin cassette was knocked into the stop codon of the DBH gene, replacing 4 bps after the last exon. B) PCR genotyping of neomycin selected ES cell clones. Targeted knock-in was determined using PCR primers that spanned from within FLPo to outside each homology arm. Amplification of a band indicates targeting. Shown are the results from a 10:1 px330_DBH_sgRNA2:targeting vector molar ratio.
Fig 3.
Analysis of DBH-FLPo sgRNA off-target sites.
No mutations were seen in the top 5 potential off-target sites. A) List of top 10 potential off-target sites as determined by the Optimized CRISPR tool, base pair mismatches, and location in the genome. B-F) Sequence chromatograms of each off-target site, showing the correct sequence for each of the 3 selected clones that were injected into blastocysts.
Fig 4.
Analysis of DBH-p2a-FLPo sgRNA off-target sites.
No mutations were seen in the top 5 potential off-target sites. A) List of top 10 potential off-target sites as determined by the Optimized CRISPR tool, base pair mismatches, and location in the genome. B-F) Sequence chromatograms of each off-target site, showing the correct sequence for each of the 3 selected clones that were injected into blastocysts.
Table 2.
Interbreeding of heterozygous DBH-FLPo and DBH-p2a-FLPo mice suggests that DBH-FLPo is a recessive loss-of-function mutation while DBH-p2a-FLPo is not.
Heterozygous DBH-FLPo mice were interbred and no homozygotes were seen among surviving weaned pups. Heterozygous DBH-p2a-FLPo mice were interbred and multiple homozygotes were seen among surviving pups.
Fig 5.
Expression of DBH-FLPo recombinase in brainstem catecholaminergic populations.
Expression of EGFP in adult DBH-FLPo; RC::FEE animals in brainstem and mid-brain catecholaminergic populations. 30 μm brain sections are stained with tyrosine hydroxylase (TH, red). EGFP co-expresses with TH in all brainstem populations but is not expressed in midbrain populations. Scale bar represents 100 μm. VTA = Ventral Tegmental Area; SN = Substantia Nigra.
Fig 6.
Expression of DBH-p2a-FLPo recombinase in brainstem catecholaminergic populations.
Expression of EGFP in adult DBH-p2a-FLPo; RC::FEE animals in brainstem catecholaminergic populations. 30 μm brain sections are stained with tyrosine hydroxylase (TH, red). EGFP co-expresses with TH in all brainstem populations but is not expressed in midbrain populations. Scale bar represents 100 μm. VTA = Ventral Tegmental Area; SN = Substantia Nigra.
Table 3.
Cell count analysis shows that nearly all cells in brainstem catecholaminergic populations in DBH-FLPo; RC::FEE and DBH-p2a-FLPo; RC::FEE mice co-express EGFP and tyrosine hydroxylase (TH).
Cells from ten random images from 3 mice per line were manually counted for co-expression of EGFP and TH.