TY - JOUR
T1 - Stable and unstable transgene integration sites in the human genome
T2 - extinction of the Green Fluorescent Protein transgene in K562 cells
AU - Migliaccio, Anna Rita
AU - Bengra, Chikh
AU - Ling, Jianhua
AU - Pi, Wenhu
AU - Li, Chunhua
AU - Zeng, Shan
AU - Keskintepe, Meral
AU - Whitney, Barry
AU - Sanchez, Massimo
AU - Migliaccio, Giovanni
AU - Tuan, Dorothy
N1 - Funding Information:
We thank Dr M. Farrell for the XIG mGFP plasmid, G. Alespeiti for initial FACS sorting and analysis, Dr K. Satya-Prakash for karyotype analysis, G. Oblak and H. Soliman for assistance with electronic graphics, Dr D. Miller for helpful discussion, and Drs K. Lanclos and D. Levin for critical review of the manuscript. The work was supported in part by funds from Piogetto finalizzato ACRO (A.R.M.), Biotec and Piano Saugue (G.M.) and NIH grants HL 39948, 48374 and 62308 (D.T.).
PY - 2000/10/3
Y1 - 2000/10/3
N2 - In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human β-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites. Copyright (C) 2000 Elsevier Science B.V.
AB - In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human β-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites. Copyright (C) 2000 Elsevier Science B.V.
KW - Competitive PCR
KW - Drug and FACS selections
KW - HS3 and CMV enhancers
KW - Long-term cell culture
KW - Southern blots
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U2 - 10.1016/S0378-1119(00)00353-X
DO - 10.1016/S0378-1119(00)00353-X
M3 - Article
C2 - 11054549
AN - SCOPUS:0034601704
SN - 0378-1119
VL - 256
SP - 197
EP - 214
JO - Gene
JF - Gene
IS - 1-2
ER -