Abstract
Kunming mice are the most widely used outbred colony in China. Differences in biological characters and drug reactions among different populations have been observed when using Kunming mice. But the molecular genetic profiles of Kunming mice and the extent of genetic differentiation among populations are unclear. Fifteen microsatellite markers were screened by a fluorescence-based semi-automated genotyping method for the two main populations of Kunming mice from Beijing (BJ) and Shanghai (SH) in China. The observed number of alleles, effective number of alleles, observed heterozygosity, unbiased expected heterozygosity and Shannon information index were used to estimate the genetic variation within the populations. A total of 89 alleles were detected in the two populations, with two to 12 at each locus, and the mean unbiased expected heterozygosity was 0.5724, which implies that there is abundant genetic variation in the populations of Kunming mice. Population differentiation was shown by shared alleles, F-statistics, Nei genetic distance and Nei genetic identity. In population BJ and population SH, respectively, only 35 of 61 and 35 of 63 alleles were shared by both. The Fst per locus varied from 0.0131 (D2Mit30) to 0.5697 (D7Mit281) and the average Fst of all loci was 0.1433, which indicates moderate genetic differentiation between the two Kunming mouse populations. The differences were also observed by Nei's [Genetic distance between populations. Am Nat 1972;
Kunming mice are the most commonly used outbred mouse line in China. Generally, it is believed that the original colony of Kunming mice in China was started from Swiss mice brought to Kunming, China, from the Indian Haffkine Institute in 1944, then it was dispersed to researchers and commercial dealers nationwide. Thereafter, these mice are called Kunming mice. Because of these animals' high disease resistance, good adaptive capacity, high breeding coefficient and good survival rate, this line has been widely utilized in pharmacological, toxicological, medicinal and biological research and testing. Thus, considerable data on Kunming mice in various fields have been accumulated, which provide valuable information and references for this and future studies.
There are many Kunming mouse populations maintained in different institutions in China, and each of them has been bred for a long time in geographical isolation. The management and the breeding programme for each population are different, and these differences have had a significant effect on the genetic character and the stability of the populations. Differences in biological characteristics and drug reactions are reported among the populations. 3–8 This leads to a lack of comparability of research results when using Kunming mice from different populations. However, a limited number of studies monitoring the genetic quality of Kunming mice were performed more than 10 years ago, using classical methods with biochemical markers, and no notable genetic differences were reported then. 9,10 Thus, the assessment of the genetic diversity and differences in populations of Kunming mice is essential in order to use them in biomedicine and government decision-making for population standardization.
Genetic evaluation for Kunming mice is currently lacking, partly due to the complexity of the genetic profiles of the outbred stock, which is defined as a restricted colony of animals with a limited increase in the inbreeding coefficient at 1% per generation and a high degree of genetic variability. 11,12 More animals and more testing loci with good polymorphisms are needed for genetic monitoring of the outbred stock. Consequently, at present, genetic testing methods and standards mainly aim at inbred strains by the government standard, and there are no standard genetic detecting methods and programmes for the outbred stock. So the molecular genetic profiles of Kunming mice and the extent of genetic differentiation among populations are unclear.
The present research is a part of the national project on the Study on Standard and Systems for Quality Detection of Laboratory Animal by the Ministry of Science and Technology of China. Microsatellite DNA, also named short tandem repeats, is more or less distributed throughout the eukaryotic genome by tandem repeats of 1–6 bp and is considered to be a valuable DNA marker for the genetic testing of laboratory animals because of its abundant loci, good polymorphisms, ease and stability of testing and Mendelian codominant inheritance. Therefore, in the present study, 15 microsatellite markers were screened by a fluorescence-based semi-automated genotyping method for the populations of Kunming mice from Beijing (BJ) and Shanghai (SH), the result of which was the first report on the molecular genetic profiles of the outbred Kunming mice. Population BJ was from the Rodent Laboratory Animal Resources of China (LARC), and population SH was from LARC, the Shanghai branch. So these animals were core populations of Kunming mice in China. We compared the relative genetic diversity and evaluated the genetic differentiation between the two populations. The results should provide data and references to eliminate differences among populations of Kunming mice and make them a more reliable animal model.
Materials and methods
Sampling and DNA isolation
Thirty mice were obtained from the population of Kunming mice maintained at the Rodent Laboratory Animal Resources (BJ), and 20 mice were obtained from the population of Kunming mice from the Rodent Laboratory Animal Resources, SH branch. Four BALB/cJ mice obtained from the Rodent Laboratory Animal Resources were used as controls. Animal breeding and tissue collection were performed in accordance with China's laws on animal experimentation and the Guide to the Care and Use of Laboratory Animals. Animals were housed in laminar flow hoods in an environmentally controlled animal facility and were fed standard rodent chow and water ad libitum. Total DNA was extracted from liver tissue according to the previous methods of our laboratory, 13 and the extracts were stored at −70°C for future use.
Polymerase chain reaction amplification and microsatellite genotyping
Fifteen microsatellite loci (D2Mit30, D2Nds3, D3Mit15, D3Mit22, D3Mit51, D5Mit48, D6Mit15, D6Mit102, D7Mit281, D7Nds1, D10Mit180, D11Mit4, D12Nds2, D13Mit3 and D13Mit88) were employed based on good polymorphisms having been reported. Primer sequences of the microsatellite loci were used according to the Mouse Locus List (
The polymerase chain reaction (PCR) conditions were optimized according to the referenced protocols. PCR amplifications were performed in aliquots of 15 μL volumes containing 2.1 μL genomic DNA (20 ng/μL), 1.50 μL PCR buffer (10 ×), 1.50 μL MgCl2 (25 mmol/L), 1.50 μL dNTP (each for 2.5 mmol/L), 0.9 μL of each primer (5 μmol/L) and 0.6 U Taq DNA polymerase. PCR were carried out as follows: an initial incubation at 95°C for 12 min, followed by 10 cycles, each consisting of 30 s at 94°C, 30 s at 53°C, 45 s at 72°C, followed by 30 cycles, each consisting of 30 s at 89°C, 30 s at 53°C, 45 s at 72°C, a final extension at 72°C for 10 min and then incubation at 4°C.
PCR products of 2 μL were mixed with 12 μL buffer (size standard: deionized formamide = 1:25), then analysed with an ABI PRISM® 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Product sizes were calculated and genotyped by an ABI PRISM® GeneScan 3.7 (Applied Biosystems) and ABI PRISM Genotyper® 3.7 (Applied Biosystems).
Data analysis
For each locus and population and across populations, commonly derived statistics from the microsatellite genotypic data viz, allele frequencies, observed number of alleles (No), effective number of alleles (Ne), 14 observed heterozygosity (Ho), expected heterozygosity (Nei's unbiased heterozygosity, He) 2 and Shannon information index were estimated. Nei genetic identity, genetic distance 1,2 and F-statistics 14 were also calculated to evaluate the level of population subdivision. The calculations were performed with the POPGENE software package version 1.32. 15 Gst' were calculated with a Microsatellite Analyzer 4.05. 16
Hardy-Weinberg equilibrium (HWE) expectations for genotype frequencies, and their associated probabilities, were calculated with exact tests by using the complete enumeration or the Markov chain methods depending on the number of alleles at each locus as implemented in the program package GENEPOP version 3.4 17,18 at default settings (10,000 dememorization steps, 100 batches and 5000 iterations per batch). Score statistics (U tests) were used to specifically assess heterozygote deficiency and excess, and also by using the Markov chain method at default settings. Two estimates of Fis, Weir and Cockerham's 19 estimate and Robertson and Hill's 20 estimate were calculated to estimate the inbreeding coefficient.
Results
Genetic variation across populations
In the present study, genetic polymorphisms in 50 individuals belonging to two Kunming mouse populations were analysed with 15 microsatellite loci. Complete lists of the allelic frequencies for each population are given in Table 1, with BALB/cJ mice as controls. The levels of genetic variation and related parameters at each locus for all the populations combined are summarized in Table 2. The following provides a summary of the results.
Allele frequencies at 15 microsatellite loci in two Kunming mouse populations
BJ: Beijing; SH: Shanghai
The genetic variation of 15 microsatellite loci across two populations, including the number of alleles observed (No), the number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), F-statistics (Fit, Fis and Fst) and Shannon's information index (I)
A fairly typical amount of polymorphisms for the two populations was discernible from the allele frequency data. All of the 15 loci were polymorphic and a total of 89 alleles were detected across the 15 loci. The numbers of alleles at each locus ranged from two (D10Mit180 and D13Mit88) to 12 (D6Mit102), with an average of 5.9333. The effective allele number ranged from 1.2295 (D10Mit180) to 7.7399 (D6Mit102), with an average of 3.1083; the observed heterozygosity (Ho) ranged from 0.0400 (D6Mit15) to 0.5833 (D13Mit3), with an average of 0.3510; the expected heterozygosity (He) ranged from 0.1866 (D10Mit180) to 0.8708 (D6Mit102), with an average of 0.5724; and the Shannon information index ranged from 0.3341 (D10Mit180) to 1.9384 (D3Mit51), with an average of 1.1602 (Table 2). All individuals of the BALB/cJ inbred line were homozygotes with the same allele for all 15 loci.
Genetic variation within populations
As shown in Table 3, the genetic variety index in the populations BJ and SH was calculated, respectively. In population BJ, 13 of 15 loci were polymorphic and two loci (D7Mit281 and D10Mit180) were monomorphic, and in population SH, 14 of 15 loci were polymorphic and one locus (D13Mit88) was monomorphic. In population BJ, 61 alleles were found altogether, ranging from one (D7Mit281 and D10Mit180) to nine (D3Mit22) at each locus, with an average of 4.0667, and 63 alleles were found in population SH, ranging from one (D13Mit88) to 10 (D3Mit51) at each locus, with an average of 4.2000. The mean observed and effective microsatellite alleles, Ho and He and the Shannon information index for population SH were all slightly greater than for population BJ. Therefore, slight differences were observed in the overall genetic variety between population BJ and population SH.
Microsatellite alleles (No: observed; Ne: effective), heterozygosity (Ho: observed, He: expected), Shannon's information index (I) in two Kunming mouse populations
Genetic differentiation between populations
Population differentiation was studied by shared alleles, F-statistics (Fit, Fis and Fst), Nei's genetic distance and Gst' between the two populations. A total of 89 alleles were detected across the 15 loci in the two populations, but only 35 alleles, 39.33% of total, existed in both populations, viz, were shared by both of them. The Fst per locus varied from 0.0131 (D2Mit30) to 0.5697 (D7Mit281) and the average Fst of all loci was 0.1433, which implied that a 14.33% genetic variation existed between the populations (Table 2). Nei's 1 genetic identity and genetic distance between the two populations were 0.6712 and 0.3987, and Nei's 2 unbiased genetic identity and genetic distance measures were 0.6783 and 0.3881, respectively. The Gst' per locus varied from −0.0547 (D2Nds3) to 0.8679 (D6Mit102) and the Gst' overall for the loci was 0.4035.
Hardy-Weinberg expectation
We compared genotype frequencies at each locus with Hardy-Weinberg expectations for each population. The P values for HWE, heterozygote deficiency and heterozygote excess and inbreeding estimates (Fis) per locus are shown in Table 4. In population BJ, except for two monomorphic loci (D7Mit281 and D10Mit180), seven of 13 loci (D2Nds3, D3Mit22, D3Mit51, D5Mit48, D6Mit15, D6Mit102, D7Nds1) exhibited significant deviations (P < 0.05) from HWE, with all loci displaying significant deficiencies in the heterozygotes (P < 0.05). In population SH, except for one monomorphic loci (D13Mit88), eight of 14 loci (D2Nds3, D3Mit22, D3Mit51, D6Mit15, D6Mit102, D11Mit4, D12Nds2, D13Mit3) exhibited significant deviations (P < 0.05), six of which exhibited significant deficiencies in the heterozygotes (P < 0.05), except for D2Nds3 and D13Mit3. The corresponding values of Fis for each locus and each population are shown.
Exact test probability of Hardy-Weinberg equilibrium (HWE), U-test probability of heterozygote deficiency and heterozygote excess, and inbreeding estimates (Fis)
ht. def.: heterozygote deficiency; ht. exc.: heterozygote excess; W&C: Weir and Cockerham's estimate; R&H: Robertson and Hill's estimate
*Exact test probability of HWE
† U-test probability of heterozygote deficiency
‡ U-test probability of heterozygote excess, ±SE was calculated when Markov chain methods were used
Discussion
Genetic profiles of Kunming mice
This is the first report of the molecular genetic profiles of Kunming mice. Despite the fact that Kunming mice have been widely utilized in biomedicine for more than 60 years in China, only limited genetic studies have been performed, and these were more than 10 years ago. The genetic quality of the outbred stock has been ignored compared with the laboratory animals made up of inbred strains. But most mice used in toxicological or pharmacological research are obtained from outbred stocks, mainly Kunming mice in China. So the genetic quality of the outbred stock is important for researchers using them, especially for toxicological or pharmacological research. In the present study, the molecular genetic profile of two core populations of Kunming mice from LARC was initially defined by 15 microsatellites. Distribution of gene frequency for all loci was obtained, and the relative genetic diversity was evaluated for each population.
The results imply that there is abundant genetic variation in populations of Kunming mice. All microsatellite loci were polymorphic for the 50 individuals of the two Kunming mouse populations, with the mean number of alleles being 5.9333 for each locus. The Ho and He values were 0.3510 and 0.5724, respectively. These values are comparable to Yu's study on house mice in Taiwan, 21 which suggested that a mean of 3.17–8.50 alleles were found at six microsatellite loci, and the expected heterozygosity varied between 0.35 and 0.83. A previous study comparing genetic profiles of outbred with wild rats suggested that heterozygosity in Rj:SD, Crl:WIST and wild rats were 0.27 ± 0.19, 0.41 ± 0.19 and 0.35 ± 0.21, respectively. 22 Thus, Kunming mice possess considerable genetic variability and is a suitable outbred stock for the purposes of toxicological or pharmacological research, etc.
More polymorphisms were also tested in outbred stocks with microsatellites than previously with allozymes. Generally up to three alleles were found at each locus with previous genetic testing of the mouse with allozymes, and a low heterozygosity was obtained. 23,24 Consequently, microsatellites are a more suitable marker for genetic monitoring of the outbred stock with genetic heterozygosity, as more detailed genetic profiles can be described according to the greater number of alleles at each locus, and sufficient loci can thus be selected for extensive distribution in the genome, which are suitable for analysis, and essential molecular genetic profiles can be compiled using microsatellites as a DNA marker. Otherwise, single-nucleotide polymorphisms (SNPs) can be also employed as a prospective method for genetic monitoring of the outbred stock because of their good reproducibility, high throughput, automation and low cost. More than eight million SNPs have been found in the mouse genome, and a variety of SNP genotyping approaches have been developed for genetic detecting of mice. 25,26
The polymorphisms in the present study may be influenced by the preselection of these loci as well as the different breeding schemes within the population. The 15 loci used in the present study were selected based on available references and our preliminary experiment results, and certain loci at which unfavourable results were obtained, e.g. unstable or excessive null alleles, were eliminated for the purpose of an accurate analysis. Only good polymorphic and reproducible loci were adopted for analysis. The effective population sizes were approximately 400 for both populations. For production efficiency as well as conservation of housing space, random mating, in which only males were selected as breeders in one mouse rack and only females were selected as breeders in the other mouse rack, and with polygamous mating, one male versus two females in each cage, were used for population BJ. For population SH, the circular group mating system, 27 with eight to 10 groups and 20 monogamous pairs in each cage within each group, was used.
Genetic differentiation between the Beijing and Shanghai populations
The results show that slightly more polymorphisms were detected in population SH than in population BJ. Nevertheless, the indices for genetic diversity, observed number of alleles, HO, He and Shannon information index were all lower in each population than across populations. This is a result of the genetic differences between the two populations. Alleles possessed by each population and the distribution within the population are different, which raises the issue of genetic diversity when the animals are combined as a whole population.
Both populations belonged to the Kunming mouse outbred stock, but only 35 of 89 alleles across populations, or 35 of 61 and 35 of 63 alleles in each population, were shared by both. This indicates that population subdivisions existed within each population, which were also evaluated by Fst and Nei's genetic identity and genetic distance.
Fst values can be used to determine the level of genetic differentiation among populations. According to Wright, 28 there were four qualitative guidelines for the interpretation of Fst and genetic differentiation: 0–0.05 for little, 0.05–0.15 for moderate, 0.15–0.25 for large and above 0.25 for very large genetic differentiation. Based on these guidelines, the mean Fst value between the populations BJ and SH in the present study fell within the range for moderate genetic differentiation between the two Kunming mouse populations. There was an extreme heterogeneity of Fst values among the loci scored. As Fst was highly sensitive to the variability of the locus, the adjusted differentiation measure proposed by Hedrick, 29 Gst', was also used. From the result, the Fst was proportional to the Gst' value at most loci, so the heterogeneity of Fst values could be attributed to the different variability of the loci for most cases, with exceptions at some loci, D10Mit180 and D6Mit102, where large differences existed between Fst and Gst' values.
The differences were also observed by Nei's 1 genetic distance (0.3987) and Nei's 2 unbiased measures of genetic distance (0.3881) estimates of subdivision. Nei's 1 genetic distances of Swiss and wild mouse populations ranged from 0.019 to 0.081. 24 Therefore, the genetic distance between the two populations was considerable, there being a low genetic identity observed between them.
Hardy-Weinberg expectation
From the results, approximately half the loci (7/13 and 8/14) of each population exhibited significant deviations from HWE, and most deviations were caused by a deficiency of heterozygous individuals, as determined by using a scoring test. The potential presence of null alleles that can also cause the deficiency in heterozygotes could not be completely ruled out, but the inbreeding effect is indicated by significant Fis values (Table 4). Deviation from HWE seems to be a frequent problem in outbred laboratory animals. 22 Different procedures to avoid inbreeding were used in the populations, but no obviously different Hardy-Weinberg expectation was observed between them. Accordingly, it is important to develop a deliberate procedure for the maximum avoidance of inbreeding by eliminating other factors influencing genetic variation and genetic stability, such as unconscious selection, etc., in the course of the propagation of Kunming mice.
This study reports the molecular genetic profiles of two main Kunming mouse populations by means of microsatellite analysis. It shows that moderate polymorphisms exist in the Kunming outbred mouse, which makes it a suitable laboratory model for biomedicine. However, considerable genetic differentiation exists between populations because of geographic isolation and long-term closed-off propagation within each population. Thus, effective comparisons of experimental results would not be easy to obtain using different Kunming mouse populations. As Kunming mice are the most widely used outbred mice in China, genetic profiles of these animals need to be unified and standardized. Intercrossing among the different main populations of Kunming mice could be an effective way to achieve this, but loss of genetic diversity must be avoided during the course of any such project. This research on Kunming mouse genetic diversity and differences should assist in developing a national plan for the unification and standardization of the populations of Kunming mice.
Furthermore, for stabilizing a reunited breeding stock of Kunming mice in future, a specific management procedure should be formulated and performed, taking into account the need for maximum avoidance of inbreeding, random genetic drift and unconscious selection. The optimal and suitable mating procedure and population size should be determined by genetic monitoring in the course of propagation. The reunited new outbred stock of Kunming mice could be renamed as LARC: Kunming, which would be the nucleus colony of future Kunming mice. LARC could then provide breeders for production colonies from the nucleus colony.
Footnotes
ACKNOWLEDGEMENTS
This project was sponsored by the scientific research condition work mission project of the Ministry of Science and Technology of China (2003), Chongqing Natural Science Foundation of China (2004–2006), National Basic Research Program of China (No. 2007CB513007) and Key Technologies R & D Program of China (No. 2006BAK02A03).