Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression

被引:33
作者
Ray, S
Paulmurugan, R
Hildebrandt, I
Iyer, M
Wu, L
Carey, M
Gambhir, SS
机构
[1] Stanford Univ, Sch Med, James H Clarke Ctr, Dept Radiol, Stanford, CA 94305 USA
[2] Univ Calif Los Angeles, Sch Med, Crump Inst Mol Imaging, Los Angeles, CA 90095 USA
[3] Univ Calif Los Angeles, Sch Med, Dept Urol, Los Angeles, CA 90095 USA
[4] Univ Calif Los Angeles, Sch Med, Dept Biol Chem, Los Angeles, CA 90095 USA
[5] Univ Calif Los Angeles, Sch Med, Dept Mol & Med Pharmacol, Los Angeles, CA 90095 USA
[6] Univ Calif Los Angeles, Sch Med, Jonsson Comprehens Canc Ctr, Los Angeles, CA 90095 USA
[7] Univ Calif Los Angeles, Sch Med, Dept Biomath, Los Angeles, CA 90095 USA
关键词
D O I
10.1089/1043034041361271
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification ( TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.
引用
收藏
页码:681 / 690
页数:10
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