Expression of coagulation factor IX (Christmas factor) in human hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer.
OBJECTIVE
In this study, we compared production of recombinant human factor IX by HepG2 cells transduced with a cytomegalovirus (CMV) promoter-controlled factor IX vector to endogenous production of factor IX by non-transduced primary rat hepatocytes.


RESULTS
Northern analysis showed 2.8 kb transcripts corresponding to the known size of factor IX mRNA in primary hepatocyte preparations and vector factor IX transcripts of the expected sizes in transduced HepG2 cell preparations. Factor IX produced by transduced HepG2 cells was completely inhibited by a monospecific antibody against human factor IX. Western analysis showed that recombinant factor IX migrated to the region of native plasma factor IX at 56 Kd. Production of biologically active factor IX by transduced HepG2 cells was 20-fold greater than that by nontransduced primary hepatocytes.


CONCLUSIONS
These data indicate that transduced HepG2 cells transcribe, synthesize, and secrete authentic factor IX, and that these genetically engineered cells secrete significantly greater amounts of factor IX than do nontransduced primary hepatocytes. Studies are in progress to determine the effect of hepatocyte mitogens on production of factor IX in transduced HepG2 cells and primary hepatocytes.
Division of Hematology-Oncology, Children's Hospital of Los Angeles, CA 90054-0700.
E M
EM
Gordon
H
H
Tang
R L
RL
Salazar
D B
DB
Kohn
D000818:Animals; D000941:Antigens / Q000097:blood; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002478:Cells, Cultured; D005164:Factor IX / Q000096:biosynthesis / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D000941:Antigens; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005164:Factor IX