Transfection of a human α-(1,3)fucosyltransferase gene into Chinese hamster ovary cells: Complications arise from activation of endogenous α-(1,3)fucosyltransferases

Barry Potvin, Ravindra Kumar, Daniel R. Howard, Pamela Stanley

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Abstract

In order to isolate a human gene encoding an α-(1,3)fucosyltransferase (α-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing α-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of α-(1,3)Fuc-T action. CHO cells do not express α-(1,3)Fuc-T activity but contain at least two, silent α-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the α-(1,3)Fuc-T activity of HL-60 cells by the letter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an α-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like α-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human α-(1,3)Fuc-T gene had been transfected. However, in all transaction experiments, isolates that expressed α-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO α-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human α-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.

Original languageEnglish (US)
Pages (from-to)1615-1622
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number3
StatePublished - Jan 25 1990

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galactoside 3-fucosyltransferase
Cricetulus
Transfection
Ovary
Genes
Chemical activation
Cells
HL-60 Cells
DNA
Fetuins
Glycosyltransferases
Gene encoding
Fucose
Enzymes
Experiments
Carbohydrates

ASJC Scopus subject areas

  • Biochemistry

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Transfection of a human α-(1,3)fucosyltransferase gene into Chinese hamster ovary cells : Complications arise from activation of endogenous α-(1,3)fucosyltransferases. / Potvin, Barry; Kumar, Ravindra; Howard, Daniel R.; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 265, No. 3, 25.01.1990, p. 1615-1622.

Research output: Contribution to journalArticle

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abstract = "In order to isolate a human gene encoding an α-(1,3)fucosyltransferase (α-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing α-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of α-(1,3)Fuc-T action. CHO cells do not express α-(1,3)Fuc-T activity but contain at least two, silent α-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the α-(1,3)Fuc-T activity of HL-60 cells by the letter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an α-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like α-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human α-(1,3)Fuc-T gene had been transfected. However, in all transaction experiments, isolates that expressed α-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO α-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human α-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.",
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