PMID- 9002671
OWN - NLM
STAT- MEDLINE
DCOM- 19970326
LR  - 20190512
IS  - 0964-6906 (Print)
IS  - 0964-6906 (Linking)
VI  - 6
IP  - 1
DP  - 1997 Jan
TI  - Functional human CFTR produced by stable Chinese hamster ovary cell lines derived 
      using yeast artificial chromosomes.
PG  - 59-68
AB  - The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a 
      transmembrane protein (CFTR) which functions in part as a cyclic adenosine 
      monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled 
      temporally and cell specifically by mechanisms that are poorly understood. 
      Insight into CFTR regulation could be facilitated by the successful introduction 
      of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To 
      this end, we have introduced two different CFTR-containing yeast artificial 
      chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. 
      Clonal cell lines containing human CFTR were identified by PCR, and the genetic 
      and functional analyses of one clone containing each YAC are described. 
      Integration of the human CFTR-containing YACs into the CHO genome at a unique 
      site in each cell line was demonstrated by fluorescence in situ hybridization 
      (FISH). Southern blot analysis suggested that on the order of one copy of human 
      CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis 
      suggested that CFTR remained grossly intact. Northern analysis showed 
      full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation 
      with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride 
      secretion in response to cAMP indicated the functional nature of the human CFTR. 
      This study provides several novel results including: (i) functional human CFTR 
      can be expressed from these YACs; (ii) CHO cells are a permissive environment for 
      expression of human CFTR; (iii) the level of human CFTR expression in CHO cells 
      is unexpectedly high given the lack of endogenous CFTR production; and (iv) the 
      suggestion by Fiber-FISH of CFTR integrity correlates with functional gene 
      expression. These YACs and the cell lines derived from them should be useful 
      tools for the study of CFTR expression.
FAU - Mogayzel, P J Jr
AU  - Mogayzel PJ Jr
AD  - Laboratories of Gene Transfer, National Center for Human Genome Research, 
      National Institutes of Health, Bethesda, Maryland 20892, USA.
FAU - Henning, K A
AU  - Henning KA
FAU - Bittner, M L
AU  - Bittner ML
FAU - Novotny, E A
AU  - Novotny EA
FAU - Schwiebert, E M
AU  - Schwiebert EM
FAU - Guggino, W B
AU  - Guggino WB
FAU - Jiang, Y
AU  - Jiang Y
FAU - Rosenfeld, M A
AU  - Rosenfeld MA
LA  - eng
GR  - DK 48977/DK/NIDDK NIH HHS/United States
GR  - HL 47122/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Hum Mol Genet
JT  - Human molecular genetics
JID - 9208958
RN  - 0 (CFTR protein, human)
RN  - 0 (RNA, Messenger)
RN  - 126880-72-6 (Cystic Fibrosis Transmembrane Conductance Regulator)
SB  - IM
MH  - Animals
MH  - CHO Cells
MH  - *Chromosomes, Artificial, Yeast
MH  - Cricetinae
MH  - Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/metabolism
MH  - *Gene Expression
MH  - Humans
MH  - RNA, Messenger
MH  - Transfection
EDAT- 1997/01/01 00:00
MHDA- 2001/03/28 10:01
CRDT- 1997/01/01 00:00
PHST- 1997/01/01 00:00 [pubmed]
PHST- 2001/03/28 10:01 [medline]
PHST- 1997/01/01 00:00 [entrez]
AID - dda020 [pii]
AID - 10.1093/hmg/6.1.59 [doi]
PST - ppublish
SO  - Hum Mol Genet. 1997 Jan;6(1):59-68. doi: 10.1093/hmg/6.1.59.