Lentiviral vectors with amplified beta cell-specific gene expression.
An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA, USA.
K L
KL
Shaw
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E
Pais
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S
Ge
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C
Hardee
D
D
Skelton
R P
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Hollis
G M
GM
Crooks
D B
DB
Kohn
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D009928:Organ Specificity; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation / Q000235:genetics*
D007328:Insulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010735:Phosphoglycerate Kinase