Experiment: Validating Gene Editing Reporter 10 (GER10) Mouse Model in Heterozygous Blastocysts
PI: Stephen A Murray, PhD
Description: WT female mouse and homozygous male mouse containing the Gene Editing Reporter 10 (GER10) reproter driven by the CAG promoter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Adenine Base Editor RNP complex and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for Venus (GFP) fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced to determine efficiency of base changes at the target region. 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, 174. Total blast analyzed by Sanger sequencing, 112.
Editing Assay:
- 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.
- Reporter activation by ABE repair is calculated as the percent of blasts that express Venus out of the total blasts imaged.
Parent Project: The Jackson Laboratory Gene Editing Testing Center (JAX-GETC) - GER10 small animal reporter, submission 1
Download: Submitted files
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Results |
| Editing Efficiency | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Condition | Cell Type | Editor | Model | Target Locus | Guide | Signal | % of blasts with Venus activation | % of blasts with edits | Image |
| ABE | ABE7.10 | GER10 Heterozygous Blastocyst | Venus* Q81X | GER10-ABE-REV |
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| Validation of Gene Editing Reporter 10 (GER10) 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 Venus fluorescence and scored for fluorescence signal. |
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