Confirmation of a G nucleotide deletion in the Cola-2 gene of mice with the osteogenesis imperfecta mutation.
Confirmation of a G nucleotide deletion in the Cola-2 gene of mice with the osteogenesis imperfecta mutation.
复制标题
证实具有成骨不全突变的小鼠 Cola-2 基因中的 G 核苷酸缺失。
DOI：
10.1006/geno.1994.1141
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发表时间：
1994
期刊：
影响因子：
4.4
通讯作者：
Shapiro,JR
中科院分区：
文献类型：
--
作者：
McBrideJr,DJ;Shapiro,JR
Osteogenesis imperfecta (OI) is a heritable disease in humans caused by mutations in the COL1A1 or COL1A2 genes that encode type l procollagen (4). Recently, mice with natu-rally occurring skeletal disease associated with a cDNA G de-Metion (termed oim) that encodes the C-propeptide of proce2 (I) were described (2). Our inspection of human (1) and mouse cDNA nucleotide sequences demonstrated a high degree of sequence identity in this region. Comparison of the oirn and wildtype murine cDNAs with human COL1A2 suggested that the G deletion was 6 bp downstream from a splice site. This report of genomic nucleotide sequence confirms that the oim mutation is in the murine proa2 (I) collagen gene, that affected animals are homozygous for the mutation, and that heterozygotes at this locus have alleles that code for both wildtype and oim mhNA. In addition, this report validates allele-specific oligonucleotides that will be important tools for use with this animal model.Genomic DNA from a wildtype mouse and mice hornozygous and heterozygous for the oim rmutation were arplified by PCR techniques (9) in a 50-ul reaction volume that contained (0.5 M each primer, 1X PCR buffer, 200 u M each dNTP, and 1.5 U Ampli" Taq (Perkin-Elmer-Cetus, Norwalk, CT). The upstream (sense) oligonucleotide was 5'-TCCAAGGAAATG-GCAACTCAGCTC-3". The downstream (anti-sense) oligonu-cleotide was 5'-GAAACAGACGGGGCCAAC-3'." I'his oligonu-cleotide pair, predicted to generate a 357-bp cDNA fragment from total RNA using the RT-PCR technique, yielded an approximate 1.2-kb genomic PCR product by 1% agarose gel analysis. Direct cycle sequencing of each genomic PCR product was performed using a murine C-propeptide anti-sense oligonucleotide (5'-GAAACAGACGGGGCCAACC-3'). Analysis of the nucleotide sequence demonstrated three distinct nu-cleotide sequences for each of the mice studied and confirmed their individual presumed genotypes based on SDS-PAGE analysis of tissue collagen samples. Direct nucleotide sequenc-ing of the anti-sense strand of the putative oirn/oim mouse demonstrated a C (G in the sense strand) deletion previously identified in the cDNA of affected mice that was present in the
