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Title: Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice

Abstract

Adenine phosphoribosyltransferase (APRT: EC 2.4.2.7), a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficientmore » animals.« less

Authors:
; ;  [1]
  1. Indiana Univ., School of Medicine, Indianapolis, IN (United States) [and others
Publication Date:
OSTI Identifier:
133965
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; CNN: Grant DK38185; TRN: 95:005313-0700
Resource Type:
Journal Article
Journal Name:
American Journal of Human Genetics
Additional Journal Information:
Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; ENZYMES; ENZYME ACTIVITY; GENES; PATIENTS; UROGENITAL SYSTEM DISEASES; HEREDITARY DISEASES; BIOLOGICAL MODELS; MICE; CHIMERAS; PHENOTYPE; ANIMAL BREEDING; ADENINES; RECESSIVE MUTATIONS; BIOLOGICAL MARKERS; CLONING

Citation Formats

Engle, S.J., Chen, J., and Tischfield, J.A. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice. United States: N. p., 1994. Web.
Engle, S.J., Chen, J., & Tischfield, J.A. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice. United States.
Engle, S.J., Chen, J., and Tischfield, J.A. Thu . "Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice". United States.
@article{osti_133965,
title = {Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice},
author = {Engle, S.J. and Chen, J. and Tischfield, J.A.},
abstractNote = {Adenine phosphoribosyltransferase (APRT: EC 2.4.2.7), a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficient animals.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {1994},
month = {9}
}