The tkNeo gene, but not the pgkPuro gene, can influence the ability of the beta-globin LCR to enhance and confer position-independent expression onto the beta-globin gene.
Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.
Mei
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Q
Centre Hospitalier de l'Université de Montréal/Institut du cancer de Montréal, Montreal, Quebec, H2L 4M1, Canada.
Kothary
R
R
Wall
L
L
D000818:Animals; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D019901:Locus Control Region; D051379:Mice; D008822:Mice, Transgenic; D009355:Neomycin / Q000494:pharmacology*; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D010957:Plasmids; D011691:Puromycin / Q000494:pharmacology*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D011691:Puromycin; D005914:Globins; D013937:Thymidine Kinase; D010735:Phosphoglycerate Kinase; D009355:Neomycin