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Effect of doxycycline‐regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant chinese hamster ovary cells
Author(s) -
Chung Joo Young,
Lim Seung Wook,
Hong Yeon Joo,
Hwang Sun Ok,
Lee Gyun Min
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10919
Subject(s) - chinese hamster ovary cell , thrombopoietin , calreticulin , microbiology and biotechnology , chemistry , western blot , cell culture , recombinant dna , cell growth , calnexin , biology , endoplasmic reticulum , biochemistry , stem cell , gene , genetics , haematopoiesis
In an attempt to increase the specific thrombopoietin (TPO) productivity ( q TPO ) of recombinant Chinese hamster ovary (rCHO) cells (CHO‐TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q TPO was investigated. To control both CNX and CRT expression levels simultaneously, the Tet‐Off system was first introduced in CHO‐TPO cells, and stable Tet‐Off cells (TPO‐Tet‐Off) were screened by luciferase assay. The doxycycline‐regulated CNX and CRT expression system in rCHO cells (TPO‐CNX/CRT) was established by cotransfection of CNX and CRT expression vector and pTK‐Hyg vector into TPO‐Tet‐Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO‐CNX/CRT cells could be tightly controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 μg/mL doxycyline), a 2.9‐fold increase in CNX expression and a 2.8‐fold increase in CRT expression were obtained in the absence of doxycycline. This, in turn, resulted in a 1.9‐fold increase in q TPO , not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q TPO of rCHO cells. © 2004 Wiley Periodicals, Inc.