Increment of hFIX expression with endogenous intron 1 in vitro

摘要

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 GlNaCi'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/pKG5i'IX, 151 ng/10~6 cells/24h; PA317/GlNaCi'IX, 308 ng/10~6 cells/24 h; C2C12/G1 NaCi'IX, 186 ng/10~6 cells/24 h; RSF/GlNaCi'IX, 1929 ng/10~6 cells/24 h; HSF/GlNaCi'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 GlNaCi'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 bio-activity. PCR detection of HT/GlNaCi'IXR cells infectedwith PA317/GlNaCi'IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron 1-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.