Quantitative RT-PCR to evaluate in vivo expression of multiple transgenes using a common intron.
Biotechniques
; 27(3): 566-70, 572-4, 1999 Sep.
Article
in En
| MEDLINE
| ID: mdl-10489616
ABSTRACT
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Introns
/
Gene Expression
/
Transgenes
/
Reverse Transcriptase Polymerase Chain Reaction
Type of study:
Diagnostic_studies
Limits:
Animals
Language:
En
Journal:
Biotechniques
Year:
1999
Document type:
Article