PMID- 10417224
OWN - NLM
STAT- MEDLINE
DCOM- 19990928
LR  - 20131121
IS  - 0006-3592 (Print)
IS  - 0006-3592 (Linking)
VI  - 64
IP  - 6
DP  - 1999 Sep 20
TI  - Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of 
      dihydrofolate reductase-mediated gene amplification and their stability in the 
      absence of selective pressure.
PG  - 741-9
AB  - Previously, the highest producing (HP) recombinant CHO subclones isolated at 
      various methotrexate (MTX) levels showed different antibody production stability 
      during long-term culture, although they were clonally derived from CS13 
      transformant. In this study, genetic basis for their difference in antibody 
      production stability was investigated using southern blot hybridization and 
      fluorescence in situ hybridization (FISH) techniques. Southern analysis of HP 
      subclones revealed that light-chain (LC) and heavy-chain (HC) cDNAs were located 
      closely within 23 kb on an amplification unit, and the configuration of LC and HC 
      cDNAs within this amplification unit was not disrupted during long-term culture 
      in the absence of MTX. However, when LC and HC genes were localized on the 
      metaphase chromosomes of HP subclones using FISH, the amplified sequences were 
      present as an extended array on diverse marker chromosomes. HP subclones selected 
      at higher MTX level had more kinds of marker chromosomes. CS13*-002 isolated at 
      0.02 microM MTX had only one marker chromosome (m002), whereas CS13*-1.0 isolated 
      at 1 microM MTX had five different ones (m10A, m10B, m10C, m10D, and m10E). Each 
      marker chromosome showed different fate during long-term culture of HP subclones 
      in the absence of MTX, resulting in different degrees of stability among the HP 
      subclones. The m10A and m10B remained unchanged, whereas the others disappeared 
      or evolved to variants with shortened amplified arrays. The cells containing 
      stable marker chromosomes constituted dominant subpopulations in CS13*-1.0, and 
      thereby CS13*-1.0 became most stable in regard to antibody production during 
      long-term culture. Furthermore, our dual-color FISH showed that the telomeric 
      ends of amplified arrays on the stable marker chromosomes were always surrounded 
      by (TTAGGG)(n) sequences, indicating that (TTAGGG)(n) sequences are closely 
      related to the stability and evolution of amplified sequences. Taken together, 
      our data show that the assessment of genotypic stability of amplified CHO cells 
      is a prerequisite for understanding their production stability during long-term 
      culture in the absence of selection pressure.
CI  - Copyright 1999 John Wiley & Sons, Inc.
FAU - Kim, S J
AU  - Kim SJ
AD  - Department of Biological Sciences, Korea Advanced Institute of Science and 
      Technology 373-1, Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea.
FAU - Lee, G M
AU  - Lee GM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biotechnol Bioeng
JT  - Biotechnology and bioengineering
JID - 7502021
RN  - 0 (Antibodies)
RN  - 0 (DNA, Complementary)
RN  - 0 (Genetic Markers)
RN  - 0 (Immunoglobulin Heavy Chains)
RN  - 0 (Immunoglobulin Light Chains)
RN  - 0 (Recombinant Proteins)
RN  - EC 1.5.1.3 (Tetrahydrofolate Dehydrogenase)
RN  - YL5FZ2Y5U1 (Methotrexate)
SB  - IM
MH  - Animals
MH  - Antibodies/genetics/*metabolism
MH  - Base Sequence
MH  - Blotting, Southern
MH  - CHO Cells/drug effects/*physiology
MH  - Cells, Cultured
MH  - Cricetinae
MH  - DNA, Complementary
MH  - *Gene Amplification
MH  - Genetic Markers
MH  - Immunoglobulin Heavy Chains/genetics/metabolism
MH  - Immunoglobulin Light Chains/genetics/metabolism
MH  - In Situ Hybridization, Fluorescence
MH  - Methotrexate/pharmacology
MH  - Recombinant Proteins/genetics/*metabolism
MH  - Selection, Genetic
MH  - Tetrahydrofolate Dehydrogenase/*genetics/metabolism
MH  - Transfection
EDAT- 1999/07/23 00:00
MHDA- 1999/07/23 00:01
CRDT- 1999/07/23 00:00
PHST- 1999/07/23 00:00 [pubmed]
PHST- 1999/07/23 00:01 [medline]
PHST- 1999/07/23 00:00 [entrez]
AID - 10.1002/(SICI)1097-0290(19990920)64:6<741::AID-BIT14>3.0.CO;2-X [pii]
PST - ppublish
SO  - Biotechnol Bioeng. 1999 Sep 20;64(6):741-9.