Construction of a chimeric gene that effectively co-expresses the human nerve growth factor receptor and cytosine deaminase genes

V. A. Lewis, N. Blake, Paul J Orchard

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Abstract

The ability to provide positive and negative selection utilizing a single gene can prove advantageous in providing a 'suicide gene' function while allowing a means to directly identify and/or select cells expressing the negative selectable element. Cytosine deaminase (CD) converts the relatively non-toxic pro-drug 5-fluorocytosine (5-FC) to the cytotoxic 5fluorouracil (5-FU). We designed a chimeric gene containing the extra-cellular and transmembrane domain of the human nerve growth factor receptor (NGFR) to provide a cell surface marker detectable by flow cytometry, and linked it to the coding sequence of the E. coli CD gene with a [gly4,ser]2 linker. A retrovirus was constructed using this chimeric NG/ CD gene, and heterogeneous populations of transduced cells isolated by neomycin resistance. The expression of NGFR was determined using flow cytometry, while CD function was tested using an MTS cytotoxicity assay in 5-FC. NIH-3T3 cells and CEM (human T-cells) transduced with the NG/CD gene or the wild-type counterparts were compared. Flow cytometric analysis revealed in repeated experiments that the expression of NGFR in cells expressing the chimeric gene was comparable to its wild type counterpart. The LD50 to 5FC for CEM cells transduced with the wild type CD gene was determined to be 30 μg/ml while the LD50 for cells expressing the chimeric NG/CD was 10 μg/ml. Similar results were obtained when NIH-3T3 cells transduced with the NG/CD or wild-type CD genes were compared. We conclude that the use of a NG/CD chimeric gene may prove useful as a 'suicide gene'providing co-expression of positive (NGFR) and negative (CD) functions. This strategy ensures the expression of the functional gene of interest (CD) in cells identified as expressing the positive selectable marker, as both functions are provided by the same gene product.

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART II
StatePublished - Dec 1 2000

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