Quantitative RT-PCR to evaluate in vivo expression of multiple transgenes using a common intron.
An assay measuring RNA expression levels of a gene-encoded therapeutic must distinguish between endogenous mRNA and mRNA transcribed from the transgene. Specificity for the delivered transgene is especially critical when the treatment involves genes that are expressed in the target tissue. To facilitate uniform detection of transgene RNA without interference from endogenous mRNA, we have engineered expression vectors that include a 5' untranslated region (5' UTR) containing a synthetic intron (PGL3). The synthetic intron splice junction was the target sequence for a quantitative reverse transcription (RT)-PCR assay utilizing Taq-Man technology. In this study, we demonstrate that a quantitative RT-PCR assay designed to recognize an engineered intron splice site in the 5'UTR of expression constructs effectively measures the expression level of in vivo-delivered gene therapeutics.
Fairman
J
J
Valentis, Burlingame, CA, USA.
Roche
L
L
Pieslak
I
I
Lay
M
M
Corson
S
S
Fox
E
E
Luong
C
C
Koe
G
G
Lemos
B
B
Grove
R
R
Fradkin
L
L
Vernachio
J
J
D020121:5' Untranslated Regions; D000199:Actins / Q000235:genetics; D000818:Animals; D016228:Endothelial Growth Factors / Q000235:genetics; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics; D007438:Introns; D007700:Kinetics; D008222:Lymphokines / Q000235:genetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019076:Transgenes; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020121:5' Untranslated Regions; D000199:Actins; D016228:Endothelial Growth Factors; D008222:Lymphokines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016179:Granulocyte Colony-Stimulating Factor; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse