Cloning and expression of canine clotting factor IX cDNAin vitro mediated by retroviral vector.
Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cDNA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, G1NaCcIX (driven by hCMV promoter) and G1NaMBcIX (driven by MCK enhancer and beta-actin promoter) were constructed using the retroviral vector backbone of G1Na. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/10(6) cell/24 h (G1NaCcIX) and 211 ng/10(6) cell/V24 h (G1NaMBcIX) respectively. Those data offered a promising result for further animal study.
Gao
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Institute of Genetics, Fudan University, 200433, Shanghai, China.
Qiu
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Lu
D
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Xue
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J