Details

Autor(en) / Beteiligte

• Wang, Sam
• Petravicz, Jeremy
• Breakefield, Xandra O

Titel

64. Bioluminescence Imaging of Regulated Gene Expression Using HSV-1 Amplicon Vectors in Rodent Brain

Ist Teil von

• Molecular therapy, 2004-05, Vol.9 (S1), p.S27-S27

Ort / Verlag

Milwaukee: Elsevier Inc

Erscheinungsjahr

2004

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• For many applications, it may be necessary to regulate gene expression in a dose-dependent manner to attain the most efficacious dose of gene product. Using the pantropic HSV-1 amplicon vector, regulatory components can be incorporated into its genome and delivered in multiple copies via the concatermizeration of amplicon sequences during virion packaging. An earlier version of the regulated amplicon vector was previously shown to express dsRed when treated with 20 nM rapamycin. More notable was the basal expression level of dsRed mRNA in an uninduced state. In the present experiments, the parental HSV-1 amplicon vector has been modified to contain both an improved activation fusion protein domain (from pC 4 N2 -RH S3H/ZF3, ARIAD Pharmaceuticals), which has been reported to possess a broader induction range, and insulator elements derived from the b-globin gene (from pJC13.1, Dr. Gary Felsenfeld, NIH), which flank the inducible promoter, to minimize basal promoter expression. The inducible cassette has also been modified to express luciferase (luc) to permit real time imaging and includes a human growth hormone intron sequence (from pZ12-I-PL5, ARIAD) to promote message stability. A RSV-driven enhanced green fluorescent protein (eGFP) was incorporated for vector titration and identification of infected cells. 293T/17 cells infected at a m.o.i. = 1 resulted in up to 70% eGFP + cells, and demonstrated a graded luc response to rapamycin (0-20 nM), with no detectable luc expression in an uninduced state. Induction of luc expression was also evident after injection of vector (2 × 105 tu) into the cortex of nude mice and i.p. injection of rapamycin (1 mg/kg) 24 hr later. By bioluminescence imaging, no luc was detectable prior to rapamycin administration, and luc expression was observed four days after induction with rapamycin. Luc expression was not detectable three days later. Experiments are currently in progress to assess repeated induction, as well as to correlate bioluminescence expression with luc histology.