Experiment: Validating Gene Editing Reporter 12 (GER12) Mouse Model in Heterozygous Blastocysts
PI: Stephen A Murray, PhD
Description: WT female mouse and homozygous male mouse containing the Gene Editing Reporter 12 (GER12) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced. ICE analysis tool from Synthego was used to score edits. NOTE: Some blasts did not PCR amplify well and it could not be determined if they had edits. Total blasts analyzed expressing the fluorescent reporter, 58-69. Total blast analyzed by Sanger sequencing, 51-52.
Editing Assay:
- Reporter activation by NHEJ is calculated as the percent of blasts that express Katushka2S (RFP) out of the total blasts imaged.
- Reporter editing efficiency assessed using PCR amplification of the reporter locus in individual blasts followed by Sanger sequencing. Percent edited blasts are expressed as the number of blasts with DNA changes in the target locus divided by the total number of blasts with a scorable chromatogram.
Parent Project: The Jackson Laboratory Gene Editing Testing Center (JAX-GETC) - GER12 small animal reporter, submission 1
Download: Submitted files
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Results |
| Editing Efficiency | ||||||||
|---|---|---|---|---|---|---|---|---|
| Condition | Editor | Model | Target Locus | Guide | Signal | % of blasts with RFP activation | % of blasts with edits | Image |
| GER12-g1-FWD | Alt-R® S.p. Cas9 Nuclease V3 | GER12 Heterozygous Blastocyst | GER12 (CAG-Venus*,-Katushka2S,-luc) | GER12-g1-FWD | ||||
| GER12-g2-FWD | Alt-R® S.p. Cas9 Nuclease V3 | GER12 Heterozygous Blastocyst | GER12 (CAG-Venus*,-Katushka2S,-luc) | GER12-g2-FWD | ||||
| Validation of Gene Editing Reporter 12 (GER12) Mouse Model in Heterozygous Blastocysts |
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Zygotes were electroporated with Cas9 RNP along with guides and cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal. |
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