Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells.

F Martiniuk,A Pellicer,R Hirschhorn

doi:10.1016/s0021-9258(17)38724-0

Abstract

To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection.

Highlights

To define new methods for gene isolation exploiting We have previously defined two isozymesof human neutral mutant mammalian cells wetransformed a mutant a-glucosidase (AB and C) based upon electrophoretic and mouse cell line deficient in glucosidase I1 with total biochemicalparameters [7,8,9]
Transfection with intact mediated transient expression of the human gene for neutral phage from a human genomic DNA library re- a-glucosidase AB inthe enzyme-deficient sulted in transienet xpression of the human gene
We transformed the neutrala-glucosidase AB-deficient mouse lymphoma cell line BW 5147 PHAR 2.7 [9] with total human genomic DNA or with its ownDNA coprecipitated with calcium phosphate

Summary

MATERIALS AND METHODS

Addition of DNA coprecipitated with calcium phosphate to a-glucosidase AB[15]. BW 5147 PHA“ 2.7 is a mutant mouse cell line derived from the cell line BW 5147 by mutagenesis and selection mammalian cells results in uptakaend expression of the DNA for resistance to thelectin Phytohemagglutinin [13]. We transformed the neutrala-glucosidase AB (glucosidase 11)-deficient mouse lymphoma cell line BW 5147 PHAR 2.7 [9] with total human genomic DNA or with its ownDNA coprecipitated with calcium phosphate. In order to establish the optimum time for detecting maximal transient gene expression for neutral a-glucosidase AB, we assayed cells at various times (8-96 h) after transformation (Fig. 1).Maximum gene expression was observed at 48 h after addition of DNA with a 2.5-fold increase in enzyme activity of cells transformed with total human DNA as compared to cells transformed with control (BW 5147 PHAR2.7) DNA. DNA diformed with total human genomic DNA as compared to cells gested to completion with BarnHI and XhoI gave the same transformed with control (BW 5147 PHAR 2.7) DNA

TrEaxnpsriesnstion of Human Glucosidaw I I

Control mouse DNA

TCohtaarlo n

DISCUSSION