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Gene repair project Gene repair vectors –pRRL SFFV IL2Rg template repair –SceI codon optimization Gene Repair-Quantitative PCR In vitro S17 Stromal cell assay In vivo BM engraftment Others –Genotyping –phenotyping Alex Chang, PhD Dr. David Rawlings lab May 18, 2009
Gene repair quantitative PCR –Multiplex Taqman PCR to quantify Wild/Repaired IL2Rg DNA sequence Common IL2Rg sequence region (an internal control) Alex Chang, PhD Dr. David Rawlings lab
GR-Q-PCR Design Exon 1Exon2Exon 3Exon 4Exon 5 Exon 6Exon 7 Exon 8 Exon 6 RT Vector seq Exon 7 Exon 8 Exon 6 Wild/Repaired seq Exon 7 Exon 8 Lox TGA Exon 6 I-SceI Sce-SCID Exon 7 Exon 8 Repaired/Wild type region Internal control region IL2Rg
Exon 6 Exon 7 Exon 8 Exon 6 46 bp 41 bp Exon 8 Lox TGA Exon 6 I-SceI Exon 7 Exon 8 GR-Q-PCR does not amplify Sce-SCID sequence Exon 8 41 bp Exon 6 46 bp GR-Q-PCR Design GR-Q-PCR amplifies Wild/Repaired sequence Wild/Repaired seq Sce-SCID
Exon 6 Exon 7 Exon 8 Exon 6 46 bp 41 bp Exon 8 63% homology/3A/T 75% homology 100% homology GR-Q-PCR Design Wild/Repaired seq
Primer/probe sequences -Designed using Primer Express 3.0 Alex Chang, PhD Dr. David Rawlings lab GR-Q-PCR Design
60 o C 64 o C 62 o C 66 o C % B6/SceSCID 0% 0.20% 0.50% 1% 2% 5% 20% 50%
60 o C 64 o C 62 o C 66 o C % B6/SceSCID 0% 0.20% 0.50% 1% 2% 5% 20% 50%
60 o C 64 o C 62 o C % B6/SceSCID 0.20% 0.50% 1% 2% 5% 20% 50%
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