Analysis of microsatellite polymorphism in inbred knockout mice

Abstract

Previously, we found that the genotype of 42 out of 198 mouse microsatellite loci, which are distributed among all chromosomes except the Y chromosome, changed from monomorphism to polymorphism (CMP) in a genetically modified inbred mouse strain. In this study, we further examined whether CMP also relates to the homologous recombination in gene knockout (KO) mouse strains. The same 42 microsatellite loci were analyzed by polymerase chain reaction (PCR) in 29 KO inbred mouse strains via short tandem sequence repeat (STR) scanning and direct sequence cloning to justify microsatellite polymorphisms. The C57BL/6J and 129 mouse strains, from which these 29 KO mice were derived, were chosen as the background controls. The results indicated that 10 out of 42 (23.8%) loci showed CMP in some of these mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite CMP in strain number 9, the core sequences of the remaining 41 loci were dinucleotide repeats, and 9 out of 41 (21.95%) showed CMPs among detected mouse strains. However, 11 out of 29 (37.9%) KO mice strains were recognized as having CMPs. The popular dinucleotide motifs in CMP were (TG)(n) (50%, 2/4), followed by (GT)(n) (27.27%, 3/11) and (CA)(n) (23.08%, 3/13). The microsatellite CMP in (CT)(n) and (AG)(n) repeats were 20% (1/5). According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a deletion. Furthermore, 2 loci (D13Mit3 and D14Mit102) revealed CMP in 2 strains, and mouse strain number 9 showed CMPs in two loci (D3Mit22 and D13Mit3) simultaneously. Collectively, these results indicated that microsatellite polymorphisms were present in the examined inbred KO mice.

Figure 1. STR loci D3Mit22 (A), D3Mit278 (B), D13Mit3 (C), and DxMit172 (C) showed microsatellite polymorphisms in knockout mice number 9, 22, 2 and 9, 4 compared to wide-type C57BL/6J and 129 mice, respectively.

The allele size of wide-type C57BL/6J, 129, and knockout mice number 9 at locus D3Mit22 are 237 bp (A-a), 220 bp (A-b), and 243 bp (A-c), respectively. The allele size of wide-type C57BL/6J, 129, and knockout mice number 22 at locus D3Mit278 are 117 bp (B-a), 95 bp (B-b), and 119 bp (B-c), respectively. The allele size of wide-type C57BL/6J, 129, and knockout mice number 2 and 9 at locus D13Mit3 are 162 bp (C-a), 161 bp (C-b), 165 bp (C-c) and 165 bp (C-d), respectively. The allele size of wide-type C57BL/6J, 129, and knockout mice number 4 at locus DxMit172 are 148 bp (D-a), 148 bp (D-b), and 146 bp (D-c), respectively.

Figure 2. Cloning sequences show microsatellite polymorphism in 10 loci (from up to low is the sequence for C57BL/6J, 129, and the knockout mouse (KO), respectively).

From figure A to J shows the sequences of locus D3Mit22, D3Mit51, D3Mit278, D8Mit14, D10Mit266, D11Mit258, D12Nds2, D13Mit3, D14Mit102, DxMit172 in turns. The underlined section indicates the repeat region. In each figure through A–J, the red lines indicate thymine, black lines indicate guanine, blue lines indicate cytosine and green lines indicate adenine.