Molecular cloning of the mouse gene coding for alpha 2-macroglobulin and targeting of the gene in embryonic stem cells.
We have cloned the mouse gene coding for alpha 2-macroglobulin in overlapping lambda clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5' flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phase A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.
Umans
L
L
Center for Human Genetics, University of Leuven, Belgium.
Serneels
L
L
Hilliker
C
C
Stas
L
L
Overbergh
L
L
De Strooper
B
B
Van Leuven
F
F
Van den Berghe
H
H
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D006801:Humans; D007438:Introns; D051379:Mice; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013234:Stem Cells / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000235:genetics*
D017931:DNA Primers; D000511:alpha-Macroglobulins