动物实验,遗传,动物造模 z-bo 2020-07-24 953

　　1. Significance The genetic testing of experimental animals is mainly the genetic testing of experimental mice. Since the use of laboratory mice as research tools, people have bred many laboratory strains, and these strains are now distributed all over the world. The total number of closed populations, inbreds, congeners, recombinant inbreds and mutants is estimated to exceed 500 species. Due to the worldwide distribution of these lines, many offspring will be produced, and there are usually many potential genetic differences between offspring of the same line. Certain daughter lines can carry more or less modified genomes while retaining their original names. Therefore, experiments with the same strain may not be able to obtain the same results, and the environment and experimental conditions are usually different, but everyone knows that this is a genetic cause. I have not yet. .. The importance of genetic testing for inbred mice is to check the inbred strains according to the standards of the Mouse Standardization and Naming Committee and remove all altered alleles.

　　 The purpose of genetic testing for interracial populations is to maintain genetic stability. In other words, in order to preserve the existing genetic heterozygosity, the gene frequency ratio of all genes in the population is always kept constant.

　　 2. Causes of genetic changes

　　 (1) Reasons for genetic changes of inbred strains

　　1. Genetic pollution The genetic pollution caused by this kind of mating is not only one kind, the foreign genome is closely related. The degree of contamination of the locus crossed with inbred strains far exceeds the occurrence of genetic mutation and genetic heterozygosity, and their occurrence is an intermittent rather than a gradual accumulation change. This kind of contamination is more common in certain strains, especially when several albino strains grow together and the breeders are untrained and of poor quality. This kind of contamination occurs and the situation becomes worse.

　　2. Residual heterozygosity uses strict sibling mating, but inbred strains retain residual heterozygosity in specific locations. It is based on 20 pairs of chromosomes, the total genome length is 2500 cents (cM), there are 60 generations of siblings or inbreds, the possibility of whole genome homozygosity is greater than 99%, homozygosity is still some genes are not. Another factor worth considering is that heterozygous genotypes are better than homozygous individuals in terms of fertilization, reproduction and survival, and they are easier to reproduce and spread.

　　3. Gene mutation Modern science has proved that gene mutation is mainly due to changes in DNA sequence. This can be a specific nucleotide substitution, deletion or insertion. From the perspective of the breeding system, mutations really work only when mutations occur in individuals representing blood.

　　 (2) Reasons for genetic changes in inbred strains

　　 Similar to inbred strains, genetic contamination and genetic mutations can cause differences in inbreds, and the distribution of allele selection in inbreds is the main reason for the changes. If the relative proportions of all gene frequencies in the outbred group remain the same, then the most suitable core breeding group would require 1,000 animals. But in fact, especially in modern times, this is impossible to do. In order to control microbial contamination, people often use caesarean section and biotechnology to rebuild new populations. During the reconstruction process, the group suddenly became smaller and selfed. The coefficient must be increased, which leads to a decrease in heterozygosity. This also means that certain genes are fixed. The anchoring of different sites in different units usually leads to changes in the distribution of alleles in the same species.

　　 3. Genetic test conditions

　　 (1) Test content

　　 1. Biochemical markers (biochemical markers). 2. Immunological tests include skin transplantation, immune marker gene testing (immunegene

　　Markers) and other methods. 3. Morphological tests include mandibular measurement (mandibular measurement), chromosome marker test (cytogenetic method) and DNA polymorphism detection (DNAmarkers).

　　 4. Coatgene test.

　　 Actually, it is impossible to adopt all the above methods, but they cannot be based on a single method. It is best to comprehensively judge whether the genetic characteristics of the strains have been based on biochemical markers, morphology and immunology.

　　 (2) Test animals

　　 When conducting genetic testing, it is necessary to reduce the frequency of testing and reduce sampling to improve efficiency.

　　 1. When testing the basic group, all the parent animals of the offspring breeding mice should be tested.

　　2. When testing the production group, if the number of females is less than 100, choose 6 males and half; if the number of females exceeds 100, choose more than half. Choose ≥6% animals. Testing requires genetic quality testing at least once a year.

　　4. Genetic testing methods (1) Morphological testing methods

　　 usually refers to morphological genetic markers used for genetic testing, mainly used in hair dye genetic testing methods and mandibles. Measurement methods and other methods that can easily detect external morphological features.

　　 1. Coat color genetic test method C.C. Little conducted the first coat color genetic test in Jackson Laboratory in 1909. This observation of coat color has always been an important indicator of inbreeding quality. Hair dye gene testing can only detect whether certain genes related to hair dye are homozygous, and cannot reflect the genetic characteristics of inbred lines, and detect mutations at other sites. It's hard to do.

　　 (1) Basic principle: mouse coat color alleles A, B, C and D are located on chromosomes 2, 4, 7 and 9, respectively. The A, B, C, and D genes are dominant in the corresponding a, b, C, and d alleles, while the C gene is the epitope of other pigment genes. If there is a CC recessive gene, there is not enough phenol in the cell and the enzyme cannot synthesize pigment. Therefore, regardless of other genes that control pigment production, mice look like albinism. The relationship between mouse fur color phenotype and genotype is as follows: "ABCD-" is wild color, "A-bbC-D-" is cinnamon, "aaB-CD-" is black, and "aabbC-D-" It's Brown, --- cc--'' is albinism. Therefore, according to genetic principles, mice with recessive alleles are crossed with the tested mice, and the genotype of the tested mice can be inferred from the fur color of F1 offspring. ..

　　（2）Method

　　① It is known that pure DBA/2 mice can be used to select recessive allele mice, whose genotype is "aabbCCdd".

　　②The tested mouse may be albino mice of other colors, but the inbred strain should be homozygous for the coat color gene of the inbred strain or the genotype of the newly cultured strain. It is usually used as a test mouse for the purpose of understanding. Of course, hybrid strains can also be used. The crossed line is used as a control group to observe the separation of the coat color gene. Method (3) In the mating method, according to the same conditions, select male mice and female mice about 70 days old with known recessive genes according to 1:1 or 1. : 2 Mate by yourself, observe and record the time and data of mating and production, and analyze the results easily

　　 (3) Standards for judging results

　　①If the F1 children have the same color, a test can be performed to determine the homozygosity of the coat color gene of the selected strain

　　②The number of F1 puppies in the same litter must be 7 or more, and the number of puppies observed in each test must be 10 or more.

　　③If there are two or more F1 puppies in the same litter, the hair color indicates that the tested mouse has genetic contamination or mutation.

　　 (4) Judgment of genotype

　　① If the F1 generation has the same shell color, the F1 generation has only one shell color, so it is judged as the gene to be tested, and only one genotype is available. Make sure that the gene is homozygous, and then estimate the genotype of the mouse you are testing. For example, use DBA/2 to test the coat color genotype of BALB/c. The DBA/2 x BALB/c F1 generation is cinnamon, and the genotype is AabbCcDd. Comparing with the known DBA/2 (silver gray) aabbCCdd genotype pair, we can see that the four alleles of A, b, c and D of F1 come from BALB/c. Because it is cinnamon, it can be inferred that the BALB/c hair dye gene tested is homozygous and the genotype is AAbbccDD.

　　②If the hair color of the F1 generation is different, the genotype of the tested mouse will be determined and tested based on the difference in the hair color of the F1 generation. Since the genotype of the mouse is not pure, the gene should be placed so that it is the genotype of the mouse you are testing, not a pure animal with a genetically different genotype.

　　2. Mandibular shape is based on mouse bones. The morphology is highly hereditary and strain-specific. This marker has become one of the traditional methods for monitoring the genetic background of experimental animals.

　　 This method compares all F1 generation coat color genotypes with known genotypes, and finds high-level animal bone morphology as a method to identify strains, including bone morphology, size, and the heritability that you can use the difference. Green has been studying this issue in 1953. In the mandibular analysis method, 20 g±1 g mouse skulls are boiled for 3 minutes or longer, trypsinized at 37°C for 24 hours, and then washed with water. The mandible is placed on an L-shaped rectangular coordinate plate, and various bone marks are measured. For length and height, enter the value of each measurement point in the table and compare it with the standard confidence zone after calculation. Being in the trusted zone means that the test is passed, while outside the trusted zone means that the test is unqualified. Generally, the morphological monitoring method is easy to operate, low cost, does not require the use of special equipment, and has strong practicability but low accuracy. (2) Cytological marker detection method The cytological marker detection method is the change of animal chromosomes, including the karyotype (the number of chromosomes, structure, the presence of satellites, the location of centromeres, etc.) and bands of the chromosomes. The type (C area, N area, G area, etc.) changes. The advantage of cytological markers over morphological markers is that they can find the chromosomes or chromosomal regions of certain important genes. However, cell markers require a lot of manpower and long-term cultivation, which is very difficult. Chromosome staining is a cytogenetic technique that allows the use of C and Q band differentiation as chromosomal markers to distinguish inbred mice from mice. In most inbred lines of rats and mice, all metaphase chromosomes have C-band material, and the C-band regions of homologous chromosomes have the same size. Different inbred lines have different C-band materials. A valuable method for detecting sub-row variation and sources of confusion. Ling Lihua et al. reported that the C-band chromosomes of T739 mice and their parents were studied. The C-band polymorphism indicated that T739 and 615 mice were inbred mice. (3) Biochemical marker detection method Biochemical marker gene detection is a routine method used to routinely detect the genetic purity of inbred animals. Inbred mice selected 10 chromosomes and 13 biochemical sites in inbred rats, and rats selected 9 biochemical sites as biochemical markers for genetic testing. Cellulose acetate membrane electrophoresis technology is usually used to detect biochemical marker genes. Find the specimen to be tested (serum, kidney homogenate, etc.), perform electrophoresis, and then develop the color. The resulting electrophoresis profile is then analyzed to determine the genotype of each test site, and these genotypes are compared with the standard gene profile of each strain to list the same and different loci. Do it. Determine the homozygosity of the test strain. It has the advantages of a well-defined locus, a simple, fast method, and a relatively economical method, but at the cost of poor accuracy.

　　 (4) Skin transplantation method

　　 Mouse skin transplantation is one of the basic methods of pure animal genetic quality control. It is simple, easy to operate and reliable. Mice and humans have a series of loci on their corresponding chromosomes. These loci form the major histocompatibility complex (MHC) or the major histocompatibility system (MHS) that controls the survival of the graft. The mouse MHC (H-2 complex located on chromosome 17) determines the main histocompatibility antigen in mice (equivalent to the human HLA system). There are 56 known mouse histocompatibility loci, such as

　　 H-1, H-2, H-3...H-56. Since histocompatibility genes are dominant, they can be expressed separately in heterozygotes. The antigenicity of each histocompatibility site is different. Mouse H-2 locus is a strong antigen that strongly rejects allografts, and the transplanted skin is destroyed in about 11 days. The remaining H sites are weak antigens, also called minor histocompatibility antigens. These sites only delay the rejection of allografts, some of which may last up to 100 days. on.

　　 transplantation can be divided into different types:

　　 1. Autologous transplantation refers to transplantation between different parts of the same person. For example, the skin on both sides of the lumbar spine on the back of a mouse is transplanted to the left and right, but the recipient can recognize that the antigen of the transplant is his or her own, so there will be no immune response and long-term survival. 2. Allogeneic transplantation refers to transplantation between individuals of the same genus. According to genetic differences, it can be divided into:

　　 (1) Same gene transplantation (same gene): One of the characteristics of pure animals is the same genotype, which is the reason for the successful skin transplantation. The transplanted antigen of the recipient is perfectly matched with the donor because it is an important basis and rejection will not occur. (2) Allogeneic transplantation: All mice are the same, but not a strain, but their genotypes are different. The transplantation of this kind of tissue must cause a specific immune response in the recipient, and the transplantation only grows in a short time. After all, it was rejected by the body. (3) Transplantation of F1 homologous inbred lines: When transplanted into F1 mice, the F1 parent tissue may grow. Since F1 contains half of the alleles of both parents, there is no immune response, but because of the different genotypes of the parents, tissue transplantation from F1 offspring to parents was rejected.

　　 When testing the skin transplantation method, select at least four adult animals of the same sex from each strain for allogeneic skin transplantation. All successful transplants are considered qualified, and transplant rejection due to non-surgical reasons is considered unqualified. (5) Detection methods of molecular biology

　　 Since the 1980s, the technology and methods of molecular biology have developed rapidly and have become very important new research tools in many fields such as biology, medicine and agriculture. It has become. Molecular biological marker technology can directly test the changes of animal genome nucleic acid, providing a direct and objective method for genetic monitoring of inbred animals. Molecular biology labeling technology mainly includes the following categories: 1. Microsatellite labeling A microsatellite is a DNA sequence that is connected with multiple nucleotides (mainly 2-4) in series. , Its polymorphism is also called simple sequence polymorphism, short tandem repeat sequence or simple sequence length polymorphism. Microsatellites are usually flanked by conserved sequences, which can be used to design specific primers for PCR amplification of microsatellite alleles. This sequence is widely present in human and other eukaryotic genomes. High abundance, high allelic variation, high individual specificity, easy detection, shared markers, stable and reliable, and good experimental repeatability. economic. Ouyang Zhaohe et al. studied the application of microsatellite DNA polymorphism in genetic monitoring of inbred mice, and selected 16 microsatellite loci on different chromosomes of mice and used PCR technology to conduct research. The 10 inbred lines used were analyzed. The result of mouse microsatellite DNA polymorphism analysis is that 14 microsatellite DNAs show stable amplification effects, showing monomorphism among different individuals of the same strain, and showing polymorphism among different strains Sex. Microsatellite polymorphism analysis at 14 loci allows rapid and economical genetic monitoring of inbred mice. 2. Random Amplified Polymorphism DNA Random Amplified DNA Polymorphism (RAPD) is a method for detecting DNA polymorphism developed by Wiliam and Welsh et al in 1990. Use randomly synthesized non-specific oligonucleotide primers (5-10 bp) to amplify genomic DNA to obtain polymorphic DNA fragments. APD objectively reflects the differences between the genomes of each organism, and most follow the rules of Mendelian inheritance. The primers used for RAPD analysis have no boundaries between species, and there is no need to know the nucleotide sequence of the gene to be analyzed in advance. Although the stability and specificity of RAPD markers are doubted, its operation is simple, fast, economical and practical, and its polymorphism detection rate is high. In mice, RAPD markers are used to analyze the genetic distance between populations for gene linkage analysis and gene mapping. Zhang Wenyan et al. 100 primers were used to amplify samples from BALB/c mice, C57BL mice and KM mice. Nearly 90% of the primers obtained amplified bands, and 21 primers were selected to amplify 3 different mouse tissue samples. In addition, there are 13 primers that can distinguish between BALB/c and C57BL mice, and there are 8 primers that can distinguish between BALB/c and KM mice. Although it is difficult to distinguish between BALB/c and KM mice based on the coat color and appearance alone, the method can only be distinguished by observing the amplified band. Wu Kaiping used RAPD-PCR to genetically test mice and screened primers that showed polymorphism among different mouse strains. APD technology has huge potential applications in laboratory animal research, but the disadvantage is that RAPD fingerprints are not reproducible, and the comparability of differential RAPD gene markers produced in different laboratories needs to be improved. That is. 3. SNP marker SNP marker (Single Nucleotide Polymorphism) is a DNA sequence polymorphism caused by a single nucleotide change at the genome level. This is due to conversion, transversion, indels and other formats. Petkov and colleagues selected 28 SNP markers. These markers cover all mouse autosomes and X chromosomes, and can distinguish all inbred mice. The results show that this method is a fast, reliable and efficient method for gene monitoring. SNP markers are used to randomly monitor all animals in an established population. The development of SNP markers has greatly improved our ability to identify inbred backgrounds. 4. EST-labeled cDNA refers to DNA complementary to the RNA sequence. cDNA does not contain introns and can be used for subsequent sequence analysis. In 1983, short cDNA sequences were discovered for gene identification; in 1991, Adams et al. proposed the concept of "EST" when they started high-throughput cDNA sequencing. Since then, Expressed Sequence Tag (EST) technology has been extensively developed worldwide. The continuous development of methods such as high-speed automatic sequencing and DNA chip sequencing has made it possible to sequence the genomes of certain organisms. EST marker technology is widely used, especially in the research of functional genomics, making it the most effective shortcut for genetic research. In 1993, the National Center for Biotechnology Information (NCBI) established a special EST database (EST, dbEST database) to store and collect all EST data. With the rapid increase in the number of ESTs in various databases, EST markers have become basic tools for biological research, discovery of new genes, construction of gene linkage maps, genetic resource resource analysis and comparative genomics research. widely used. 5. The difference in mtDNA sequence of mitochondrial DNA genetic markers can be directly measured by restriction enzyme site analysis and mitochondrial genome sequencing. Restriction site analysis uses restriction endonucleases to identify and cut specific bases in mtDNA, and then electrophoresis the cut fragments to analyze and compare the addition or deletion of restriction fragments. The method can be further divided into restriction endonuclease mapping, restriction endonuclease fragment length polymorphism (RFLP), probe and PCR-RFLP. Among them, PCR-RFLP is now widely used in mtDNA analysis to avoid cumbersome mtDNA purification.

　　RFLP analysis of mtDNA is very simple, fast, and rich in detection of variation, but in the end it is an indirect method to detect DNA sequence variation. The direct sequencing method determines the complete sequence of mtDNA or the sequence of specific gene fragments to compare the differences between different species or individuals, and directly detect changes in DNA nucleotide positions, thereby providing the most complete data. Will be more reliable. Early sequencing methods were considered expensive and unsuitable for studying the evolution of genes in large populations and samples. Since the invention of PCR and the emergence of "universal primers", direct sequencing methods have become more and more popular in animal mtDNA research. Morphological testing, cytological marker testing, biochemical marker testing, skin transplantation are traditional monitoring methods and indirect genetic testing methods. These principles mainly use changes in phenotypic traits to infer corresponding genetic changes. The above-mentioned phenotypic characteristics as genetic markers have shortcomings such as low accuracy, sensitivity to external environmental factors, and lack of genetic stability. Most markers are found in all chromosomes and loci of inbred animals. Unable to overwrite. Methods (such as chromosome banding technology and immune genetic markers) are complicated to operate, which brings a lot of inconvenience to the genetic monitoring of inbred animals. Molecular biology markers basically refer to specific DNA fragments that can reflect specific differences in the genome of a single organism or population. Compared with phenotypic markers, DNA molecular marker technology can detect individuals at different developmental stages, different tissues, organs and even cells, and is not affected by the environment or restricted by gene expression. Stable; the biological function is neutral and easy to operate. It can detect multiple sites on the genome at the same time, is rich in polymorphism, and is more accurate and reliable than traditional markers. Current molecular labeling technology has disadvantages such as complex and harsh conditions for DNA extraction, amplification reaction conditions, difficulty in controlling the stability of amplified products, and high experimental costs, which limit the application of this technology. However, actual applications must follow the principles of accuracy, effectiveness, simplicity and economy. With the further development of science and technology, molecular biology technology will continue to be perfected and perfected, providing a broader scope for the development and management of our laboratory animals, and is considered to have a breakthrough effect.

　　3. Genetic test conditions\n(1) Test content\n 1. Biochemical markers (biochemical markers). 2. Used for immunological examination, skin transplantation, immunological marker gene detection (immunogen gene marker) and other methods. 3. Morphological examination includes mandibular measurement (mandibular measurement), chromosome marker detection (cytogenetic method) and DNA polymorphism detection (DNAmarkers). 4. Coatgene test. \r\nIt is actually impossible to use all the above methods, but they cannot be based on a single method. It is best to comprehensively judge the genetic characteristics of strains based on biochemical markers, morphology and immunology. \r\n(2) Experimental animals\nWhen conducting genetic testing, it is necessary to minimize the frequency of testing and reduce sampling to improve efficiency. \r\n 1. When testing the basic group, all parent animals of the offspring breeding mice should be tested. \r\n 2. When testing the production group, if the number of females is less than 100, select 6 males, half, and if the number of females exceeds 100, select more than half. Choose animals with 6% or more. Testing requires genetic quality testing at least once a year. \r\n4. Genetic testing (1) Morphological testing\n usually refers to morphological genetic markers used for genetic testing, and genetic testing for hair dyes and mandibles will be completed. Measurement methods and other methods that can easily detect external morphological features. \r\n 1. Coat color genetic test method C.C. Little conducted the first coat color genetic test in Jackson Laboratory in 1909. This observation of coat color has always been an important indicator of inbreeding quality. The hair dye genetic test can only detect whether a specific gene related to the hair dye is homozygous. It cannot reflect the genetic characteristics of inbred lines, nor can it detect mutations elsewhere.(1) Basic principle: Mouse coat color alleles A, B, C and D are located on chromosomes 2, 4, 7 and 9, respectively. The A, B, C, and D genes are dominant in the corresponding a, b, C, and d alleles, while the C gene is the epitope of other pigment genes. The CC recessive gene makes mice look chlorotic, and has nothing to do with other genes that regulate pigment production, because the cell lacks phenol and the enzyme cannot synthesize pigment. The relationship between mouse fur color phenotype and genotype is as follows: "ABCD-" is wild color, "A-bbC-D-" is cinnamon, "aB-CD-" is black, and "aabbC-D-" Brown---cc--" is albinism. Therefore, according to genetic principles, mice carrying recessive alleles are crossed with the tested mice, and the genotype of the tested mice can be inferred from the fur color of F1 offspring (2) Methods\n①It is known that pure DBA/2 mice can be used to select recessive allele mice, and their genotype is "aabbCCdd".\r\n②The tested mice can be albinism with other colors Mouse, but the inbred line must be homozygous for the coat color gene of the inbred line or the genotype of the newly cultured strain. It is usually used as a test mouse for understanding purposes. Of course, hybrid strains can also be used. Crossed lines are used as The control group was used to observe the separation of the coat color gene. Method (3) In the mating method, according to the same conditions, select about 70-day-old male and female mice with known recessive genes according to 1:1 or 1. :2 proceed Self-mating, observe and record the time and data of mating and production, and easily analyze the results\n(3) The criteria for judging the results\n①F1 children of the same color can be tested to determine the homozygosity of the selected strain’s coat color gene .\N②In each study, F1 puppies have at least 7 litters and at least 10 puppies. Must have. ③If you have more than one F1 puppies in your litter, the color of the coat indicates your mouse Has been genetically contaminated or mutated.\r\n(4) Judgment of genotype\n①If the F1 generation has the same shell color, the F1 generation has only one shell color, so it is judged as the gene to be tested, and There is only one genotype. Make sure that the gene is homozygous, and then estimate the genotype of the mouse you are testing. For example, use DBA/2 to test the coat color genotype of BALB/c. DBA/2 x BALB/c F1 is Cinnamon, the genotype is AabbCcDd. Compared with the known DBA/2 (silver gray) aabbCCdd genotype pair, we can see that the four alleles A, b, c, and D of F1 are all from BALB/c. Because It is cinnamon, we can infer that the tested BALB/c hair dye gene is homozygous, and the genotype is AAbbccDD.\r\n②If the hair color of the F1 generation is different, it will be determined and tested according to the difference in the hair color of the F1 generation. Test the genotype of the mouse. Since the genotype of the mouse is not pure, you need to place the gene to make it the genotype of the mouse you are testing, not in a pure animal whose gene is different from the known genotype .\R\n 2. The shape of the lower jaw depends on the bones of the mouse. The morphology is highly hereditary and strain-specific. This marker has become one of the traditional methods for monitoring the genetic background of experimental animals.This method compares all F1 generation coat color genotypes with known genotypes, and provides an advanced method to identify strains that can be used for differences, such as bone morphology, size, and heritability. Find the horizontal animal bone form. Green has been studying this issue in 1953. For mandibular analysis, 20 g ± 1 g mouse skulls were boiled for at least 3 minutes, trypsinized at 37°C for 24 hours, and then washed with water. Place the mandible on the L-shaped rectangular coordinate plate and measure various bone marks. For length and height, enter the value of each measurement point into the table and compare with the standard confidence interval after calculation. Being in the trusted zone means the test passed, but outside the trusted zone means the test failed. Generally, the morphological monitoring method is easy to operate, low cost, does not require the use of special equipment, and has strong practicability but low accuracy. (2) Cytological marker detection method Cytological marker detection method refers to the change of animal chromosomes, such as karyotype (chromosome number, structure, presence of satellites, centromere position, etc.) or chromosome bands. The type (C area, N area, G area, etc.) changes. The advantage of cytological markers over morphological markers is the ability to find chromosomes or chromosomal regions of certain important genes. However, cell markers require a lot of physical labor and long-term cultivation, which makes it extremely difficult. Chromosome staining is a cytogenetic technique that allows the use of C and Q band differentiation as chromosomal markers to distinguish mice from inbred lines. In most rat and mouse inbred lines, all metaphase chromosomes have C-band material, and the C-band regions of homologous chromosomes are the same size. Different inbred lines have different C-band materials. A valuable method to detect the source of sub-row variation and confusion. Ling Lihua et al. reported that the C-band chromosomes of T739 mice and their parents were studied. The C-band polymorphism indicated that T739 and 615 mice were inbred mice. (3) Biochemical marker detection method Biochemical marker gene detection is a common method to routinely detect the genetic purity of inbred animals. Inbred mice selected 10 chromosomes and 13 biochemical sites in inbred rats, and rats selected 9 biochemical sites as biochemical markers for genetic testing. Cellulose acetate membrane electrophoresis technology is usually used to detect biochemical marker genes. Find the sample to be tested (serum, kidney homogenate, etc.) and perform electrophoresis for color development. The resulting electrophoresis profile is then analyzed to determine the genotype of each test site, and these genotypes are compared with the standard genetic profile of each strain to list the same and different loci. .. Do it to determine the homozygosity of the test strain. Its advantages are clear trajectory, simple and fast method, relatively economical, but low accuracy. \r\n(4) Skin transplantation\nMice skin transplantation is one of the basic methods of pure animal genetic quality control, and it is easy to operate and reliable. Mice and humans have a set of loci on their corresponding chromosomes that form the major histocompatibility complex (MHC) or major histocompatibility system (MHS) that controls the survival of the graft. Do it. Mouse MHC (H-2 complex on chromosome 17) determines the major histocompatibility antigen in mice (equivalent to the human HLA system). There are 56 known mouse histocompatibility loci, including:\r\n H-1, H-2, H-3...H-56. Due to the advantages of histocompatibility genes, they can Expressed separately in heterozygotes. The antigenicity of each histocompatibility site is different. Mouse H-2 locus is a strong antigen that can strongly reject allografts, and the skin after transplantation is destroyed about 11 days later. The remaining H sites are weak antigens, also called minor histocompatibility antigens. These sites only delay the rejection of allograft, some of which may last up to 100 days. on. \r\nMigration can be divided into many types. \r\n 1. Autologous transplantation is a transplantation between different parts of the same person. For example, the skin on both sides of the lumbar spine of the back of a mouse is transplanted from one side to the other, but the recipient can recognize that the antigen of the transplant is his or her own, so it can survive for a long time without generating an immune response. 2. Allogeneic transplantation is a transplantation between individuals of the same genus. According to genetic differences, it can be divided into:\n (1) Same gene transfer (same gene): One of the characteristics of pure animals is the same genotype, that is, skin transfer. Is the reason for success. The antigen of the transplant recipient matches exactly with the antigen of the donor. This is an important principle, because rejection does not occur. (2) Allogeneic transplantation: All mice are the same, but not strains, but have different genotypes. The transplantation of this type of tissue must elicit a specific immune response in the recipient, and the transplantation can only last for a short time. Finally, it was rejected by the body. (3) Transplantation of F1 homologous inbred lines: When transplanted into F1 mice, F1 parent tissue may grow. There is no immune response because F1 contains half the alleles of the two parents, but because the genotypes of the parents are different, tissue transplantation from F1 offspring to parents is rejected. \r\nWhen testing the skin transplantation method, please select at least four same-sex adult animals from each strain for allogeneic skin transplantation. All successful transplants are considered qualified, and transplant rejection due to non-surgical reasons is considered unqualified. (5) Detection methods of molecular biology\nSince the 1980s, the technology and methods of molecular biology have developed rapidly and have become very important new research tools in many fields such as biology, medicine and agriculture. it is. It has become. Molecular biological marker technology can directly test the changes of animal genome nucleic acid, providing a direct and objective method for genetic monitoring of inbred animals. Molecular biology marker technology mainly includes the following categories: 1. Microsatellite marker Microsatellite is a DNA sequence in which multiple nucleotides (mainly 2-4) are connected in series. This polymorphism is also called simple sequence polymorphism, short tandem repeat sequence or simple sequence length polymorphism. Microsatellites are usually flanked by conserved sequences, which can be used to design specific primers for PCR amplification of microsatellite alleles. This sequence is widely present in human and other eukaryotic genomes. High abundance, high allelic variation, high individual specificity, easy detection, marker sharing, stability and reliability, and excellent experimental reproducibility. economic. Ouyang Zhaohe et al. studied the application of microsatellite DNA polymorphism in genetic monitoring of inbred mice. 16 microsatellite loci were selected on different chromosomes of mice, and PCR technology was used to analyze the used 10 inbred lines. The results of mouse microsatellite DNA polymorphism analysis showed that 14 microsatellite DNAs had a stable amplification effect, showing monomorphism among different individuals of the same strain, and showing polymorphism among different strains. Microsatellite polymorphism analysis at 14 loci allows rapid and economical genetic monitoring of inbred mice. 2. Random Amplified Polymorphism DNA Random Amplified DNA Polymorphism (RAPD) is a method for detecting DNA polymorphism developed by Wiliam and Welsh in 1990. Randomly synthesized non-specific oligonucleotide primers (5-10 bp) are used to amplify genomic DNA to obtain polymorphic DNA fragments. APD objectively reflects the genomic differences of each organism, and most follow the rules of Mendelian inheritance. The primers used for RAPD analysis have no boundaries between species, and there is no need to know the nucleotide sequence of the gene to be analyzed in advance. Although the stability and specificity of RAPD markers are questionable, their operation is simple, fast, economical and practical, and their polymorphism detection rate is high. In mice, RAPD markers are used to analyze the genetic distance between populations for gene linkage analysis and gene mapping. Zhang Wenyan et al. 100 primers were used to amplify BALB/c, C57BL and KM mouse samples. Almost 90% of the primers were used to obtain amplified bands, and 21 primers were selected to amplify 3 different mouse tissue samples. In addition, there are 13 primers that can distinguish between BALB/c and C57BL mice, and there are 8 primers that can distinguish between BALB/c and KM mice. Although it is difficult to distinguish between BALB/c and KM mice only by the color and appearance of the coat, the method can only be distinguished by observing the amplified bands. Wu Kaiping used RAPD-PCR to genetically test mice to screen out primers that exhibit polymorphism among different mouse strains. APD technology has great potential in the study of experimental animals, but the disadvantage is that RAPD fingerprints are not reproducible, and the comparability of different RAPD gene markers produced in different laboratories needs to be improved. That is. That is. 3. SNP marker SNP marker (Single Nucleotide Polymorphism) is a DNA sequence polymorphism caused by a single nucleotide change at the genome level. This is due to conversion, transversion, indels and other formats. Petkov and colleagues selected 28 SNP markers. These markers cover all mouse autosomes and X chromosomes, and can distinguish all inbred mice. The results show that this method is a fast, reliable and efficient method for gene monitoring. SNP markers are used to randomly monitor all animals in an established population. The development of SNP markers has greatly improved our ability to identify inbred backgrounds. 4. EST-labeled cDNA refers to DNA complementary to RNA sequence. cDNA does not contain introns and can be used for subsequent sequence analysis. In 1983, a short cDNA sequence was discovered for gene identification; in 1991, Adams et al. proposed the concept of "EST" when they started high-throughput cDNA sequencing. Since then, Expressed Sequence Tag (EST) technology has been extensively developed worldwide. The continuous development of methods such as high-speed automatic sequencing and DNA chip sequencing has made it possible to sequence the genomes of specific organisms. EST marker technology is widely used, especially in functional genomics research, it represents the most effective shortcut for genetic research. In 1993, the National Center for Biotechnology Information (NCBI) established a special EST database (EST, dbEST database) to store and collect all EST data. With the rapid increase in the number of ESTs in various databases, EST markers have become the basic tools for biological research, new gene discovery, gene linkage map construction, genetic resource resource analysis and comparative genomics research. It is widely used. 5. The difference in mtDNA sequence of mitochondrial DNA gene markers can be directly measured by restriction enzyme cut site analysis and mitochondrial genome sequencing. In restriction site analysis, restriction endonucleases are used to identify and cut specific bases in mtDNA, perform electrophoresis on the cut fragments, and analyze and compare the addition or deletion of restriction fragments. The method can be further divided into restriction endonuclease mapping, restriction endonuclease fragment length polymorphism (RFLP), probe and PCR-RFLP. Among them, PCR-RFLP is widely used in mtDNA analysis to avoid cumbersome mtDNA purification. \r\nAlthough mtDNAFLP analysis is very simple, fast and has rich mutation detection functions, it is ultimately an indirect method to detect DNA sequence mutations. The direct sequencing method determines the complete sequence of mtDNA or specific gene fragments to compare the differences between different species or individuals, and directly detects changes in DNA nucleotide positions to obtain the most complete data. provide. Will be more reliable. Early sequencing methods were considered expensive and unsuitable for studying the evolution of genes in large populations and samples.自从PCR的发明和“通用引物”的出现以来，直接测序已在动物mtDNA研究中越来越流行。形态学测试，细胞学标志物测试，生化标志物测试和皮肤移植是传统的监测方法和间接基因测试方法。这些原则主要使用表型性状的变化来推断相应的遗传变化。上述作为遗传标记的表型特征具有诸如准确性低，对外部环境因素的敏感性低以及缺乏遗传稳定性的缺点。在近交动物的所有染色体和基因座上都发现了大多数标记。它不能被覆盖。方法（如染色体显带技术和免疫遗传标记）操作复杂，给自交系动物的遗传监测带来许多不便。分子生物学标记物基本上是指可以反映单个生物体或种群的基因组中特定差异的特定DNA片段。与表型标记相比，DNA分子标记技术可以检测处于不同发育阶段，不同组织，器官甚至细胞的个体，不受环境影响，并且不受基因表达的限制。稳定，生物学中性且易于操作。它可以同时检测基因组上的多个位点，具有丰富的多态性，并且比传统标记更准确，更可靠。当前的分子标记技术具有限制其适用性的缺点，包括DNA提取的复杂且苛刻的条件，扩增反应条件，难以控制扩增产物的稳定性以及高昂的实验成本。但是，实际应用必须遵循准确性，有效性，简单性和经济性的原则。随着科学技术的进一步发展，分子生物学技术已经并将继续完善，将为实验动物的开发和管理提供更广泛的范围，并有望带来突破性的效果。\r\nV。如何分析，判断和处理基因测试结果Since the invention of PCR and the emergence of "universal primers", direct sequencing has become more and more popular in animal mtDNA research. Morphological testing, cytological marker testing, biochemical marker testing and skin transplantation are traditional monitoring methods and indirect genetic testing methods. These principles mainly use changes in phenotypic traits to infer corresponding genetic changes. The aforementioned phenotypic characteristics as genetic markers have disadvantages such as low accuracy, low sensitivity to external environmental factors, and lack of genetic stability. Most markers are found on all chromosomes and loci of inbred animals. It cannot be overwritten. Methods (such as chromosome banding technology and immune genetic markers) are complicated to operate, which brings a lot of inconvenience to the genetic monitoring of inbred animals. Molecular biology markers basically refer to specific DNA fragments that can reflect specific differences in the genome of a single organism or population. Compared with phenotypic markers, DNA molecular marker technology can detect individuals at different developmental stages, different tissues, organs and even cells, and is not affected by the environment and is not restricted by gene expression. Stable, biologically neutral and easy to operate. It can detect multiple sites on the genome at the same time, is rich in polymorphism, and is more accurate and reliable than traditional markers. The current molecular labeling technology has disadvantages that limit its applicability, including complex and harsh conditions for DNA extraction, amplification reaction conditions, difficulty in controlling the stability of amplified products, and high experimental costs. However, actual applications must follow the principles of accuracy, effectiveness, simplicity and economy. With the further development of science and technology, molecular biology technology has been and will continue to be improved, which will provide a broader scope for the development and management of laboratory animals, and is expected to bring breakthrough effects. \r\nV. How to analyze, judge and process genetic test results

　　 1. Reasons for deviation from the genetic map If the tested genetic traits match the genetic map of each strain, the genetic control information is clear. You can trust and consider strains related to breeding. Production is proceeding normally, and the genetic quality of the strain is certified. If it does not match the genetic map, it may be due to the following reasons:

　　 (1) Genetic pollution: Genetic pollution is the most common genetic variation, usually due to poor genetic control. If animal strains with the same coat color are kept and maintained in the same breeding unit, it is easy to cause false crosses between the strains. If this condition is detected as early as possible, multiple genetic markers may be observed whose characteristics, heterozygosity or other phenotypes are not consistent with the genetic characteristics studied. If discovered later, some heterozygous genes will be corrected to a single homozygous type that may be randomly present but does not match the genetic profile of the original strain. If any of the above occurs, the original strain should be eliminated, the source and quality qualified animal should be replaced with a new species, and the strain should be added again. (2) Genetic drift: Genetic drift refers to the phenomenon of random changes in gene frequency among a small number of people. Most of the changes are due to the fact that several heterozygous genes in inbred animals split before becoming homozygous, leading to the formation of sublines and branches. The test showed that all animals of this pedigree did not match the genetic spectrum of the original pedigree with 1-2 genetic markers, but they were the same in phenotype and were all homozygous. After the allogeneic transplantation test eliminates genetic contamination, this situation needs to be renamed according to the offspring and branch. (3) Gene mutation: The frequency of gene mutation in mammals is 10 ^ 5. The analysis of the test results shows that only one animal is heterozygous for a single gene marker, and whether a gene mutation has occurred should be considered. In order to confirm this, it is usually necessary to query the phenotype of animal litter or parental genetic markers based on the number of animals. If the phenotype of the genetic marker matches the genetic profile of the litter or siblings, the tested individual should be considered to have genetic variation. If the genetic mutation occurs in the parental generation, siblings in the same litter must be separated to produce an atypical or different phenotype. If during the test it is found that the phenotype of another genetic marker is inconsistent with the genetic map, whether it is heterozygous or homozygous, please consider genetic contamination and eliminate replacement immediately. Need to do this. If the gene mutation does not have a specific retention value, the entire strain can be retained even if the mutant is eliminated. In rare cases, mutants can only be reproduced as homologous inbred lines.

　　 2. Genetic test results and judgment of treatment When testing animals, heterozygous types will be detected. These animals usually need to be deleted and updated and cannot be used as inbred lines. If the mutation site is found to be homozygous, appropriate measures need to be taken. Table 1-2 shows the treatment principles established in accordance with the "China National Standards for Laboratory Animal Genetic Quality Testing of Lactation", which can usually be treated according to these principles.