REGULAR ARTICLES
Cell Research (1997)7:21-29
© 1997 SIBS, CAS All rights reserved 1001-0602/97
Increment of hFIX expression with endogenous intron 1 in vitro.
Zheng B, Qiu XY, Tan M, Xing YN, Lo D, Xue JL, Qiu XF.
Institute of Genetics, Eudan Univerisity, Shanghai
Correspondence:
Zheng B |
 Table
of Contents
Download as printable
(PDF) file
Full Text (html)file
Search Medline for articles
by:
Zheng B
|
This paper probes into the feasibility of increasing expression level of
hFIX gene with endogenous intron 1 sequence. hFIX minigene was obtained
with middle sequence truncated intron 1 inserted into the relative site
of hFIX cDNA, and plasmid vector pKG5i'IX, retroviral vector GINaCi'IX were
constructed. These vectors were transduced into target cells of PA317, C2C12,
primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts
(HSF). The expression level of mixed colonies are PA317/pKGoi'IX, 151 ng/10(6)
cells/24h; PA317/G1NaCi'IX, 308 ng/10(6) cells/24 h; C2C12/G1 NaCi'IX, 188
ng/10(5) cells/24 h; RSF/G1NaCi'IX, 1929 ng/10(5) cells/24 h; HSF/G1NaCi'IX,
1646 ng/10(6) cells/24 h. These results indicated that hFIX minigene with
intron 1 is able to increase the expression level to about 3 times of that
of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene
in the retroviral-mediated gene transfer system and refrain from intron
splicing during viral production, a retroviral vector G1NaCi'IXR with reversely
inserted hFIX minigene expression cassette was constructed. The expression
level of reverse constructor in PA317 cells was 390 ng/10(6) cells/24 h
with 79% of bioactivity. PCR detection of HT/G1NaCi'IXR cells infected with
PA317/G1NaCi'IXR supernatant confirmed the existence of intron 1 sequence.
These results suggested that expression vector with forward-inserted intron1-carrying
hFIX expression cassette can be used in directed gene transfer, but when
using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying
hFIX expression cassette should be considered.
Keywords : Intron
1, human clotting factor IX (hFIX), gene transfer, gene expression, reverse
inserted sequence. |
