Experiment: Validating Traffic Light Reporter 2 (TLR2) Mouse Model in Homozygous Blastocysts using SpCas9
PI: Stephen A Murray, PhD
Description: Male and female homozygous mice mouse containing the Traffic Light Reporter 2 (TLR2) 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 tagRFP 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, 156-193. Total blast analyzed by Sanger sequencing, 84-120.
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 NHEJ is calculated as the percent of blasts that express tagRFP out of the total blasts imaged. Approximately one third of NHEJ events are detected.
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 |
| Guide 26-1 | Alt-R® S.p. Cas9 Nuclease V3 | TLR-2 mouse homozygous blastocyst | Gt(ROSA)26Sortm12(CAG-Venus*,-TagRFP*)Rkuhn | R26-1 | ||||
| Guide 26-2 | Alt-R® S.p. Cas9 Nuclease V3 | TLR-2 mouse homozygous blastocyst | Gt(ROSA)26Sortm12(CAG-Venus*,-TagRFP*)Rkuhn | R26-2 |
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| Guide 26-3 | Alt-R® S.p. Cas9 Nuclease V3 | TLR-2 mouse homozygous blastocyst | Gt(ROSA)26Sortm12(CAG-Venus*,-TagRFP*)Rkuhn | R26-3 | ||||
| Validation of Traffic Light Reporter 2 (TLR2) Mouse Model in Homozygote 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 tagRFP fluorescence and scored for fluorescence signal. |
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