Use Of Microsatellite DNA For Genetic Monitoring Of Inbred Strain Mice

Inbred strain mice is remarkable for its characteristics: homozygosity, isogenicity and stability. Using inbred strain mice can not only reduce labratoty animal numbers and experimental repetition times, but improve experiment accuracy, comparability. But there still exists possibility of genetic contamination or genetic mutation during breeding, breeding conservation and production of inbred strain mice. Therefore, strict, long-term, and timely genetic monitoring for inbred strain mice is necessary. Traditional methods, such as skin grafting, biochemical markers, immunologic methods and other methods by inferring gene changes from epitype changes, have many limits.Microsatellite DNA, comprising short tandem repeated sequences,is ubiquitous in eukaryotic genomes.It offers unusual advantages, such as abundant polymorphism, extensive distribution, large quantity, high stability,and easy to amplify.So it can directly reflect gene change at DNA level and reflect genetic profile and variant situation of genome much more generally and easily b.This method has so many merits that it will be an ideal genetic monitoring marker for inbred strain animal.In this experiment, we used 20 pair primers to study DNA polymorphism at different allelli. First, genome DNA of these mice was extracted and amplified by PCR using microsatellite primers; then these amplified products were displayed by electrophoresis on denaturing polyacrylamide gel and silver-dyed, expecting to screen microsatellite loci that have polymorphism and can be applied to monitor genetic of inbred mice.Methods: Five mice were selected from each of the seven inbred strain mice colony(DBA/2, CBA, C57, BALB/C, C3H/HeJ, FVB/NJ, YYHL mice) and one closed mice colony (Kunming mice).First, genome DNA of these mice were extracted from their brain samples by phenol-chloroform method, then amplify 20 microsatellite DNA loci (incluting:D4mit9,D5mit31,D6mit15,D6mit16,D3mit36,D7mit12,D7mit29,D8mit11,D8mit22,D8mit24,D8mit30,D9mit21,D10mit29,D11mit35,D11mit36,D12mit8,D12mit29,D12mit34,D12mit35,D13mit18) by PCR.PCR products were electrophoresed on 4-6% denaturing polyacrylamide gel, and in sequence the gel was fixed, silver-dyed and color-developed until the bands were clear. By comparison these bands of PCR products of microsatellite locus and allelic ladder, we recorded these results according the DNA electrophoresis distance on denaturing polyacrylamide gel and counted the polymorphism bands displayed on studied loci among these eight mice colony. The results were calculated by the similarity coefficient according to Lynth's method, and the similarity coefficient were analyzed.Results:1. Purity analysis of microsatellite loci: PCR products of different strain and different individual of the same strain show one single band, which indicated that these gene loci were pure.2. Comparison of microsatellites polymorphism among these inbred strain mice:(1) Genetypes of alleles and number of alleles about various strain mice: 20 pair primers could be amplified stably and efficiently. Among the 20 microsatellite loci , 16 loci(D4Mit9,D5Mit31,D6Mit15,D6Mit16,D6Mit36,D7Mit12,D8Mit24,D8Mit30,D9Mit21,D11Mit35,D11Mit36,D12Mit8,D12Mit29,D12Mit34,D12Mit35,D13Mit18) displayed polymorphism among these various strains inbred mice and the number of alleles was 2-3, in which 13 loci (D4Mit9,D5Mit31, D6Mit15,D6Mit36,D8Mit24,D8Mit30,D9Mit21,D11Mit35,D11Mit36,D12Mit29,D12Mit34,D12Mit35,D13Mit18) displayed remarkable polymorphism.(2) Similarity coefficient among different strain mice: among these inbred mice:Similarity coefficient between CBA and DBA/2, FVB/NJ and C3H/HeJ, is 0.650; between FVB/NJ, CBA and DBA/2 is 0.600; between CBA and BALB/C is 0.350.. While the similarity coefficient between closed colony Kunming mice and inbred strain BALB/C is 0.700; between Kunming mice and C3H/HeJ is 0.450;between Kunming mice and YYHL mice, DBA/2 and C57 is 0.550.3. Microsatellite polymorphism analysis among different individuals of the same inbred strain:Among 20 loci, bands of PCR products among the same strain mice on the same loci were same,which mean there is no monomorphism.Conclusions:1. Among the 20 tested loci,all inbred mice displayed one single band, indicating that these inbred mice meet the requests of inbred strain mice.2. Microsatellite DNA of inbred mice can be amplified stably,and efficiently by using PCR.3.16 microsatellite loci (D4Mit9,D5Mit31,D6Mit15,D6Mit16,D6Mit36,D7Mit12,D8Mit24,D8Mit30,D9Mit21,D11Mit35,D11Mit36,D12Mit8,D12Mit29,D12Mit34,D12Mit35,D13Mit18) out of 20 loci displayed high polymorphism in these strains, among which 13 loci(D4Mit9,D5Mit31,D6Mit15,D6Mit36,D8Mit24,D8Mit30,D9Mit21,D11Mit35,D11Mit36,D12Mit29,D12Mit34,D12Mit35,D13Mit18 )displayed remarkable polymorphism.These microsatellite loci provided one fast, convenient, and economical method for genetic monitoring on inbred mice.There was no polymorphism among these different individuals of the same strain mice.4. The similarity coefficient of these different strain mice reflect their ancestor's kindred.