Quantitative RT-PCR analysis demonstrates that synthesis of the recombinant fibrinogen is dependent on the transcription and synthesis of γ-chain

Abstract

Background: The purpose of this study was to examine the relationship between the production of secreted fibrinogen and the synthesis of γ-chain mRNA. Methods: We transfected a γ-chain expression vector into Chinese hamster ovary cells already expressing both Aα- and Bβ-chains of fibrinogen and measured fibrinogen output concentrations by ELISA. We quantified both γ-chain and Bβ-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 γ-chain mRNA ( r=0.7077, p<0.01) but not with the concentration of Bβ-chain mRNA ( r=0.0224, NS). Conclusions: These results demonstrate that the amount of recombinant fibrinogen produced in cells transfected with the γ-chain vector, also expressing normal Aα- and Bβ-chains, is dependent on the transcription of γ-chain mRNA. Namely, in this recombinant expression system using a two-step transfection procedure, γ-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.