Experiment: Validating Traffic Light Reporter 7 (TLR7) Mouse Model for NHEJ in Heterozygous Blastocysts
PI: Stephen A Murray, PhD
Description: WT female mouse and homozygous male mouse containing the Traffic Light Reporter 7 (TLR7) 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, 169. Total blast analyzed by Sanger sequencing, 117.
Editing Assay:
- Reporter activation by NHEJ is calculated as the percent of blasts that express Katushka2S (RFP) out of the total blasts imaged. Approximately one third of NHEJ events are detected.
- 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) - TLR7 small animal reporter, submission 1
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Other experiments in this project: 1
Download: Submitted files
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Results |
| Editing Efficiency | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Condition | Editor | Model | Target Locus | Guide | # blasts assessed for RFP activation | % of blasts with RFP activation | # blasts assessed by sequencing | % of blasts with edits | Image |
| RNP | Alt-R® S.p. Cas9 Nuclease V3 | TLR-7 mouse heterozygous blastocyst | C57BL/6J-Gt(ROSA)26Sor<em8(CAG-Venus*,-Katushka2S)Murr>/MurrMmjax | TLR7-g2-REV |
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| NHEJ Validation of Traffic Light Reporter 7 (TLR7) 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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