Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells

Anna Rita Migliaccio, Chikh Bengra, Jianhua Ling, Wenhu Pi, Chunhua Li, Shan Zeng, Meral Keskintepe, Barry Whitney, Massimo Sanchez, Giovanni Migliaccio, Dorothy Tuan

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)197-214
Number of pages18
JournalGene
Volume256
Issue number1-2
DOIs
StatePublished - Oct 3 2000

Fingerprint

K562 Cells
Human Genome
Green Fluorescent Proteins
Transgenes
Genes
Flow Cytometry
Locus Control Region
Genome
Globins
Gene Silencing
Cytomegalovirus
Drug Resistance
Genetic Therapy
Plasmids

Keywords

  • Competitive PCR
  • Drug and FACS selections
  • HS3 and CMV enhancers
  • Long-term cell culture
  • Southern blots

ASJC Scopus subject areas

  • Genetics

Cite this

Stable and unstable transgene integration sites in the human genome : extinction of the Green Fluorescent Protein transgene in K562 cells. / Migliaccio, Anna Rita; Bengra, Chikh; Ling, Jianhua; Pi, Wenhu; Li, Chunhua; Zeng, Shan; Keskintepe, Meral; Whitney, Barry; Sanchez, Massimo; Migliaccio, Giovanni; Tuan, Dorothy.

In: Gene, Vol. 256, No. 1-2, 03.10.2000, p. 197-214.

Research output: Contribution to journalArticle

Migliaccio, AR, Bengra, C, Ling, J, Pi, W, Li, C, Zeng, S, Keskintepe, M, Whitney, B, Sanchez, M, Migliaccio, G & Tuan, D 2000, 'Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells', Gene, vol. 256, no. 1-2, pp. 197-214. https://doi.org/10.1016/S0378-1119(00)00353-X
Migliaccio, Anna Rita ; Bengra, Chikh ; Ling, Jianhua ; Pi, Wenhu ; Li, Chunhua ; Zeng, Shan ; Keskintepe, Meral ; Whitney, Barry ; Sanchez, Massimo ; Migliaccio, Giovanni ; Tuan, Dorothy. / Stable and unstable transgene integration sites in the human genome : extinction of the Green Fluorescent Protein transgene in K562 cells. In: Gene. 2000 ; Vol. 256, No. 1-2. pp. 197-214.
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AU - Pi, Wenhu

AU - Li, Chunhua

AU - Zeng, Shan

AU - Keskintepe, Meral

AU - Whitney, Barry

AU - Sanchez, Massimo

AU - Migliaccio, Giovanni

AU - Tuan, Dorothy

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