Exploration of high exp ressions of retroviral vectors containing hF Ⅸ minigene in murine myoblast cells
10.3760/j:issn:0253-2727.2001.03.002
- VernacularTitle:含hFⅨ微小基因的杂合型逆转录病毒载体在小鼠成肌细胞中高效表达的研究
- Author:
Jun HOU
1
;
Jianmin WANG
;
Bihe MIN
Author Information
1. Changhai Hospital Second Military Medical University
- From:
Chinese Journal of Hematology
2001;22(3):121-124
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effect of different vector structure on hFⅨ protein expression in murine myoblasts in order to construct more efficient expression vector for myoblast-mediated gene therapy for hemophil ia B. Methods Three types of retroviral vectors pLMe2 Ⅸ m2 SN ( forward Me2: Me2 inserted into enhancer region of 3＇LTR in forward orientation)、pLMe2Ⅸm2SN(reversed Me2) and pLⅨ m2SN which contained intron m2 and two copies of MCK enhancer (Me2)were transferred into packaging cell line PA317. The hFⅨ expression level in selected single and mixed clones of stably transfected myoblasts were determined by ELISA and PCR. Results There was statistically significant difference in hFⅨ expression levels among the four vectors tested. The expression order was LMe2Ⅸm2SN(forward) > LMe2Ⅸm2SN(reversed) > LⅨm2SN > LⅨSN. The hFⅨ expression level of forward Me2 vector was higher than that of reversed one, because of one or two copies of reversed Me in the 5＇LTR being deleted in the genome of myoblasts, but the orientation of Me or Me2 remained unchanged. Conclusions To select a tissue-specific enhancer and optimize FⅨ minigene construction are effective methods for increasing the expression level. Forward Me2 vector can express hFⅨ more stably than the reversed one.
