title: Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells.
abstract: 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 microg/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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, Korea.
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Chung
authors.forename: Seung Wook
authors.initials: SW
authors.lastname: Lim
authors.forename: Yeon Joo
authors.initials: YJ
authors.lastname: Hong
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Hwang
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
mesh_terms: D000818:Animals; D016466:CHO Cells; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D037282:Calreticulin / Q000378:metabolism*; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D015539:Platelet Activation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D037282:Calreticulin; D011994:Recombinant Proteins; D037281:Calnexin; D013926:Thrombopoietin; D004318:Doxycycline