Quantitative analysis of the expression of a VIP transgene.
We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.
Tolentino
P J
PJ
Division of Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
Dikkes
P
P
Tsuruda
L
L
Ebert
K
K
Fink
J S
JS
Villa-Komaroff
L
L
Lamperti
E D
ED
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic; D014660:Vasoactive Intestinal Peptide / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D014660:Vasoactive Intestinal Peptide; D001616:beta-Galactosidase