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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.

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Presentation on theme: "Gene repair project Gene repair vectors –pRRL SFFV IL2Rg template repair –SceI codon optimization Gene Repair-Quantitative PCR In vitro S17 Stromal cell."— Presentation transcript:

1 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

2 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

3 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

4 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

5 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

6 Primer/probe sequences -Designed using Primer Express 3.0 Alex Chang, PhD Dr. David Rawlings lab GR-Q-PCR Design

7 60 o C 64 o C 62 o C 66 o C % B6/SceSCID 0% 0.20% 0.50% 1% 2% 5% 20% 50%

8 60 o C 64 o C 62 o C 66 o C % B6/SceSCID 0% 0.20% 0.50% 1% 2% 5% 20% 50%

9 60 o C 64 o C 62 o C % B6/SceSCID 0.20% 0.50% 1% 2% 5% 20% 50%


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