Enhanced transgene expression in embryonal carcinoma stem cells: transcription through introns and exons increases gene copy numbers and forestalls silencing.
Efficient high-level expression of transfected genes in stem cells is difficult to achieve. Using P19 embryonal carcinoma stem cells, we previously showed that cotransfection with a fragment of the Pgk-1 gene increased the number of integrated copies of genes on cotransfected plasmids and resulted in enhanced expression of these genes. Active transcription through introns and exons of the Pgk-1-derived sequence was required for elevated expression of the cotransfected reporter genes. We show here that the enhanced expression appears to be due to two effects: (1) increased ligation of plasmid DNAs prior to integration into the host genome and (2) maintenance of an active domain in the region comprising the cluster of transgenes. We found that individual clones of stably transformed cells had highly variable numbers of transgenes integrated into their genomes but the ratio of genes comprising each transgene was proportional to the ratio of genes on different plasmids in the transfection mixture. The presence of the Pgk-1 gene fragment ensured that the expression of cotransfected genes in each clone was proportional to the numbers of integrated gene copies and selection for high-level expression of one cotransfected gene ensures high-level expression of the other cotransfected genes.
McBurney
Michael W
MW
Ottawa Regional Cancer Centre, University of Ottawa, 503 Smyth Road, Ottawa K1H 1C4 Canada. michael.mcburney@orcc.on.ca
Yang
Xiaofeng
X
Jardine
Karen
K
D000818:Animals; D054278:Embryonal Carcinoma Stem Cells; D005091:Exons; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D017930:Genes, Reporter; D007438:Introns; D014411:Neoplastic Stem Cells / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013223:Statistics as Topic; D014158:Transcription, Genetic; D019076:Transgenes
D011993:Recombinant Fusion Proteins