Restricted beta-galactosidase expression of a hygromycin-lacZ gene targeted to the beta-actin locus and embryonic lethality of beta-actin mutant mice.
beta-actin is a cytoskeletal protein that is ubiquitously expressed. To exploit the regulation the beta-actin gene, a promoterless hygromycin-lacZ fusion gene with a splice acceptor was introduced into the first intron of the beta-actin locus by homologous recombination in mouse embryonic stem (ES) cells. The targeted ES cells were hygromycin resistant and expressed beta-galactosidase (beta-gal) activity. However, no beta-gal activity was detected in heterozygous embryos. In adult heterozygotes, beta-gal activity was detected only in testes. RT-PCR analysis demonstrated the presence of both beta-actin exon 1-hygromycin- and exon l-exon 2-containing transcripts in homozygous mutant embryos. LacZ-containing transcripts were detected in adult heterozygous tests and, surprisingly, in homozygous mutant embryos. These results demonstrate that the integration of the hygromycin-lacZ gene into the first intron of the beta-actin locus was not productive for the ubiquitous expression of beta-gal activity. Because this integration mimics certain types of gene trap events, it suggests that caution should be used when interpreting beta-gal expression patterns in genetic screens using gene trap strategies. In addition, mice homozygous for the beta-actin mutation developed normally up to embryonic day 8.5 (E8.5) but became growth retarded at E9.5 and subsequently died. The RT-PCR data indicate that this targeted mutation is a hypomorphic allele of beta-actin.
Shawlot
W
W
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Deng
J M
JM
Fohn
L E
LE
Behringer
R R
RR
D000199:Actins / Q000235:genetics*; D017398:Alternative Splicing; D000818:Animals; D002478:Cells, Cultured; D002934:Cinnamates; D005314:Embryonic and Fetal Development; D005091:Exons; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D005796:Genes; D017930:Genes, Reporter; D006921:Hygromycin B / Q000031:analogs & derivatives; D007763:Lac Operon; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013234:Stem Cells / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D000199:Actins; D002934:Cinnamates; D011993:Recombinant Fusion Proteins; D006921:Hygromycin B; C026273:hygromycin A; D001616:beta-Galactosidase