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

alex.chang@seattlechildrens.orgDr. 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 alex.chang@seattlechildrens.org

Dr. David Rawling’s lab

GR-Q-PCR Design

Exon 1 Exon2 Exon 3 Exon 4 Exon 5

Exon 6 Exon 7

Exon 8

Exon 6RT Vector seq Exon 7 Exon 8

Exon 6Wild/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 bpExon 8

Lox

TGA

Exon 6

I-SceI

Exon 7 Exon 8

GR-Q-PCR does not amplify Sce-SCID sequence

Exon 8 41 bpExon 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 bpExon 8

63% homology/3’A/T75% homology 100% homology

GR-Q-PCR Design

Wild/Repaired seq

GR-PCRRepPF 5'CTT GTT TGT TAC AGA GAA TCC TTC CTTGR-PCRRepPR 5'TGG TGC CAA CAG GGA TAA GCGR-PCRRepProbe FAM 5'TTT GCA CTG GAA GCT G MGBGR-PCRComPF 5'CCT TGG CAA TCT GGA TCC AAGR-PCRComPR 5'GGG ACA GGA AAT CGA AAC TTA GCGR-PCRComProbe VIC 5'CTC ACT GCC TTC CCT T MGB

Primer/probe sequences-Designed using Primer Express 3.0

Alex Chang, PhDalex.chang@seattlechildrens.org

Dr. David Rawling’s lab

GR-Q-PCR Design

60oC

64oC

62oC

66oC

% B6/SceSCID

0%

0.20%

0.50%

1%

2%

5%

20%

50%

60oC

64oC

62oC

66oC

% B6/SceSCID

0%

0.20%

0.50%

1%

2%

5%

20%

50%

% WILD/Sce-SCID y = 0.6783e-0.9254x

R2 = 0.9878

0.001

0.01

0.1

1

0 1 2 3 4 5 6 7

% WILD/Sce-SCID y = 0.2014e-0.6417x

R2 = 0.9794

0.001

0.01

0.1

1

-1.5 0.5 2.5 4.5 6.5

% WILD/Sce-SCIDy = 0.0757e-0.5508x

R2 = 0.9964

0.001

0.01

0.1

1

-3.5 -1.5 0.5 2.5 4.5 6.5

60oC

64oC

62oC

% B6/SceSCID

0.20%

0.50%

1%

2%

5%

20%

50%