[Exploration of high expressions of retroviral vectors containing hFIX minigene in murine myoblast cells].
OBJECTIVE
To explore the effect of different vector structure on hF IX protein expression in murine myoblasts in order to construct more efficient expression vector for my oblast-mediated gene therapy for hemophilia B.


METHODS
Three types of retroviral vectors pLMe2 IX m(2)SN (forward Me2: Me2 inserted into enhancer region of 3'LTR in forward orientation), pLMe2 IX m(2)SN (reversed Me2) and pL IX m(2)SN which contained intron m(2) 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 LMe2 IX m(2)SN (forward) > LMe2 IX m(2)SN (reversed) > L IXm(2)SN > L IX SN. The hF IX 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.
Hou
J
J
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Wang
J
J
Min
B
B
D000818:Animals; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D002836:Hemophilia B / Q000235:genetics* / Q000628:therapy; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000378:metabolism; D032446:Myoblasts / Q000378:metabolism / Q000821:virology*
D005164:Factor IX