Quantitative RT-PCR analysis demonstrates that synthesis of the recombinant fibrinogen is dependent on the transcription and synthesis of gamma-chain.
BACKGROUND
The purpose of this study was to examine the relationship between the production of secreted fibrinogen and the synthesis of gamma-chain mRNA.


METHODS
We transfected a gamma-chain expression vector into Chinese hamster ovary cells already expressing both Aalpha- and Bbeta-chains of fibrinogen and measured fibrinogen output concentrations by ELISA. We quantified both gamma-chain and Bbeta-chain mRNA concentrations using the recently developed TaqMan fluorogenic detection system.


RESULTS
The concentration of secreted fibrinogen into the media positively correlated with the amount of fibrinogen contained in the cell lysates. Additionally, quantitative mRNA assays revealed that the fibrinogen concentration in the cell lysates correlated well with the concentration of gamma-chain mRNA (r=0.7077, p<0.01) but not with the concentration of Bbeta-chain mRNA (r=0.0224, NS).


CONCLUSIONS
These results demonstrate that the amount of recombinant fibrinogen produced in cells transfected with the gamma-chain vector, also expressing normal Aalpha- and Bbeta-chains, is dependent on the transcription of gamma-chain mRNA. Namely, in this recombinant expression system using a two-step transfection procedure, gamma-chain synthesis is the rate-limiting factor for fibrinogen production. This quantitative method to measure mRNA may prove very useful for further in vivo analysis of fibrinogen gene transcription.
Hirota-Kawadobora
Masako
M
Central Clinical Laboratory, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
Tozuka
Minoru
M
Yamauchi
Kazuyoshi
K
Hidaka
Eiko
E
Ueno
Ichiro
I
Sugano
Mitsutoshi
M
Terasawa
Fumiko
F
Okumura
Nobuo
N
Katsuyama
Tsutomu
T
Shigematsu
Hidekazu
H
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts / Q000737:chemistry; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D021122:Protein Subunits; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D002457:Cell Extracts; D017077:Culture Media, Conditioned; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005340:Fibrinogen