PMID- 15305049
OWN - NLM
STAT- MEDLINE
DCOM- 20050214
LR  - 20061115
IS  - 1424-859X (Electronic)
IS  - 1424-8581 (Linking)
VI  - 107
IP  - 1-2
DP  - 2004
TI  - Suitability of a CMV/EGFP cassette to monitor stable expression from human 
      artificial chromosomes but not transient transfer in the cells forming viable 
      clones.
PG  - 9-13
AB  - Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC 
      constructs containing alpha satellite DNA of various human chromosomes. To 
      monitor which cells took up constructs and subsequently formed stable clones 
      under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted 
      into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). 
      Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting 
      bright green fluorescence on day 1. Areas containing such early green cells were 
      marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a 
      viable clone developed. In the remaining 40 areas, the green cells ceased 
      division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones 
      formed which did not exhibit green fluorescence during the first cell divisions, 
      but all cells of each became green around day 4-6. Fluorescence in situ 
      hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC 
      formation without integration. We conclude that transient expression of an EGFP 
      marker on HAC DNA is not a suitable means for the identification of the 
      proportion of transfected cells which are capable of forming viable clones. One 
      explanation could be that the high copy number required to consistently detect 
      transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and 
      clone formation.
CI  - Copyright 2004 S. Karger AG, Basel
FAU - Laner, A
AU  - Laner A
AD  - Medical Genetics, Childrens Hospital, Ludwig Maximilians University, Munich, 
      Germany.
FAU - Schwarz, T
AU  - Schwarz T
FAU - Christan, S
AU  - Christan S
FAU - Schindelhauer, D
AU  - Schindelhauer D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Switzerland
TA  - Cytogenet Genome Res
JT  - Cytogenetic and genome research
JID - 101142708
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Cell Line
MH  - Chromosomes, Artificial, Human/*genetics
MH  - Chromosomes, Artificial, P1 Bacteriophage/genetics
MH  - Cytomegalovirus/*genetics
MH  - Green Fluorescent Proteins/biosynthesis/*genetics
MH  - Humans
MH  - Telomere/genetics
MH  - Transfection/methods
EDAT- 2004/08/12 05:00
MHDA- 2005/02/16 09:00
CRDT- 2004/08/12 05:00
PHST- 2004/04/29 00:00 [received]
PHST- 2004/05/28 00:00 [accepted]
PHST- 2004/08/12 05:00 [pubmed]
PHST- 2005/02/16 09:00 [medline]
PHST- 2004/08/12 05:00 [entrez]
AID - 79564 [pii]
AID - 10.1159/000079564 [doi]
PST - ppublish
SO  - Cytogenet Genome Res. 2004;107(1-2):9-13. doi: 10.1159/000079564.