Culture method of immunodeficient nude mice

Abstract

The invention relates to the technical field of experimental animal culture, and discloses an immunodeficient nude mouse culture method, which comprises a genotype early-stage identification step based on the morphological characteristics of newborn suckling mouse beards, and specifically comprises the following steps: observing the beards in the mandibular and perinasal areas of a suckling mouse within 12 hours after the suckling mouse is born; if the number of the beards is less than 10, the length is less than 0.5 mm, the beards are curled and hair follicles are sparsely distributed, judging that the beards are homozygotes (nu / nu); if the number is larger than 20, the length is larger than or equal to 1.2 mm, and the seeds grow vertically and are arranged tightly, the seeds are judged to be heterozygotes (n < u / + >). According to the culture method of the immunodeficient nude mice, early-stage accurate screening and efficient culture of the homozygous nude mice can be realized through fine identification of beard forms of newborn suckling mice and milk replacement management of heterozygous breeding mice.

Description

Technical Field

[0001] The invention relates to the technical field of experimental animal cultivation, in particular to a method for cultivating immunodeficient nude mice. Background Art

[0002] The breeding of immunodeficient nude mice (nu / nu homozygous) relies on precise genetic screening. However, traditional methods lack reliable phenotypic markers for rapid identification during the neonatal period. This results in heterozygotes (nu / +) being co-housed with homozygotes until weaning, leading to the elimination of homozygotes after weaning. This results in a survival rate of only 60%-70% for homozygotes due to nutritional competition (such as uneven distribution of maternal milk) and spatial compression, especially when mating patterns employ a single male with multiple females, such as one male with two females or one male with three females. Furthermore, to prioritize the quality of homozygotes, the stress of lactation can lead to greater nutritional competition when co-housing heterozygous females for breeding, resulting in growth retardation and impacting subsequent reproductive performance. A gap in the existing technology: A rapid genetic phenotype identification system based solely on appearance in the early postnatal period, such as one to two days, has not been established for neonatal mice. In particular, there is a lack of research on the correlation between whisker development characteristics and genotype. Summary of the Invention

[0003] (1) Technical problems solved In response to the shortcomings of the existing technology, the present invention provides a method for culturing immunodeficient nude mice, which is capable of achieving early accurate screening and efficient cultivation of homozygous nude mice through the refined identification of the whisker morphology of newborn mice and the management of surrogate milk for heterozygous breeding mice, solving the core problems of low survival rate and unstable breeding mouse quality in traditional methods, increasing production, providing more high-quality animals for the market, and promoting the rapid development of biomedicine.

[0004] (2) Technical solution To achieve the above-mentioned objectives, the present invention provides the following technical solutions: a method for culturing immunodeficient nude mice, comprising an early genotype identification step based on the morphological characteristics of the whiskers of newborn suckling mice, specifically: within 12 hours after the birth of the suckling mice, the whiskers of the mandible and nasal area are observed. If the whiskers are less than 10 in number, less than 0.5 mm in length, curled, and the hair follicles are sparsely distributed, the mice are judged to be homozygous (nu / nu); if the whiskers are greater than 20 in number, ≥1.2 mm in length, grow upright, and are closely arranged, the mice are judged to be heterozygous (nu / +).

[0005] Preferably, the treatment of heterozygotes after the identification step is: euthanasia of heterozygotes within 24 hours of birth, or reservation of additional heterozygous female mice according to breeding standards.

[0006] Preferably, the reserved heterozygous female mice are subjected to intensive management of milk substitute, including: taking tail tip tissue during the weaning period (21 days of age) for PCR genotyping identification, using primers F: 5'-GTGCTGCTGCTGCTGATG-3' and R: 5'-CTGCTGCTGCTGCTGCTA-3', and amplifying a 450bp band in the heterozygote.

[0007] Preferably, the surrogate adaptation operation includes: transferring 3-day-old, 2-3g-weighted heterozygous female mice to surrogate mother cages, the surrogate mothers are heterozygous female mice or ICR female mice of the same species that gave birth at the same time, and covering the pup cages with surrogate mother bedding 24 hours in advance to contaminate them with the scent, and each cage of surrogate mothers is paired with no more than 3 pups.

[0008] Preferably, 1% whey protein powder is added to the feed during the milk replacement period to ensure that the average daily weight gain of the young mice is ≥0.3g, and the body weight reaches 9-12g at 3 weeks of age.

[0009] Preferably, the lactation management of homozygous suckling mice is: retain 3-4 homozygous suckling mice in each litter to reduce the lactation pressure of the mother mouse.

[0010] Preferably, a health monitoring step is also included: establishing a morphological profile of the whiskers of suckling mice, combined with weekly blood routine monitoring, wherein the homozygotes focus on monitoring the lymphocyte ratio, requiring CD3+T cells to be <1%, and constructing a triple monitoring system of "phenotype-genotype-immune indicators".

[0011] Preferably, the beard morphology observation is performed under a light source magnifying glass to clearly identify the number, length, curvature and hair follicle distribution of the beard.

[0012] Preferably, the selection criteria for the surrogate mothers are individuals with high milk production and strong maternal instincts.

[0013] Preferably, the immunodeficient nude mice are BALB / c background nude mice.

[0014] (3) Beneficial effects Compared with the prior art, the present invention provides a method for culturing immunodeficient nude mice, which has the following beneficial effects: 1. This method for culturing immunodeficient nude mice takes less than 3 minutes per litter to identify newborns, which means the identification time is earlier, increasing the efficiency by 90% compared to traditional genetic testing methods (which take 24 hours). Alternatively, the efficiency can be significantly improved by waiting until about 7 days of age to identify newborns.

[0015] 2. The method for culturing immunodeficient nude mice showed that after heterozygotes were eliminated, the survival rate of homozygous suckling mice at 21 days of age increased from 78% to 98% (data from the example), and the weaning weight increased by 25% (9.1g vs. 7.5g).

[0016] 3. The culture method of the immunodeficient nude mice: heterozygous female mice fed with surrogate milk have a normal estrous cycle (4-5 days) after adulthood, the age of first birth is advanced to 77 days (87 days for the traditional group), and the number of pups per litter is stable at 6-8.

[0017] 4. The culture method of this immunodeficient nude mouse maintains a homozygous ratio of 50%±3% in the offspring, which conforms to Mendel's law of inheritance and ensures the stability of continuous supply.

[0018] 5. This method of cultivating immunodeficient nude mice can eliminate heterozygotes at an early stage, thus reducing the cost of ineffective breeding by 35%. The milk substitute technology can increase the utilization rate of breeding mice by 50%. The annual cost of large-scale cultivation can be reduced by about RMB 350,000 per 10,000 mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flowchart for the management of milk surrogate for heterozygous female mice (including key nodes for genotype identification and milk surrogate adaptation).

[0020] Figure 2 Schematic diagram of phenotype-genotype association characteristics. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] See also Figure 1-2 A method for culturing immunodeficient nude mice includes an early genotype identification step based on the morphological characteristics of the whiskers of newborn mice. Specifically, within 12 hours after the birth of the mice, the whiskers on their mandible and nasal area are observed. If the whiskers are less than 10 in number, less than 0.5 mm in length, curled, and sparsely distributed with hair follicles, they are determined to be homozygous (nu / nu); if the whiskers are greater than 20 in number, ≥1.2 mm in length, grow upright, and are closely arranged, they are determined to be heterozygous (nu / +).

[0023] The treatment of heterozygotes after the identification step is: euthanasia and elimination of heterozygotes within 24 hours of birth, or reservation of additional heterozygous female mice according to the breeding standards.

[0024] The reserved heterozygous female mice were given intensive milk replacement management, including: taking tail tip tissue for PCR genotyping identification during the weaning period (21 days of age) with primers F: 5'-GTGCTGCTGCTGCTGATG-3' and R: 5'-CTGCTGCTGCTGCTGCTA-3', and a 450bp band was amplified in the heterozygotes.

[0025] The surrogate adaptation operation includes: transferring 3-day-old, 2-3g-weight heterozygous female mice to the surrogate mother cage. The surrogate mother is a heterozygous female mouse or ICR female mouse of the same species that gave birth at the same time. The pup cage is covered with surrogate mother bedding 24 hours in advance to contaminate it with the scent. Each cage of surrogate mother is paired with no more than 3 pups.

[0026] During the milk replacement period, 1% whey protein powder was added to the feed to ensure that the average daily weight gain of the young mice was ≥0.3g, and the weight reached 9-12g at 3 weeks of age.

[0027] The lactation management of homozygous puppies is: keep 3-4 homozygous puppies in each litter to reduce the lactation pressure of the mother mouse.

[0028] It also includes health monitoring steps: establishing a whisker morphology file for suckling mice, combined with weekly blood routine monitoring, among which the homozygotes focus on monitoring the lymphocyte ratio, requiring CD3+T cells to be <1%, and constructing a "phenotype-genotype-immune indicator" triple monitoring system.

[0029] The beard morphology was observed under a light source magnifying glass to clearly identify the number, length, curvature and hair follicle distribution of the beard.

[0030] The selection criteria for the surrogate mothers were individuals with high milk production and strong maternal instincts.

[0031] Immunodeficient nude mice were BALB / c background nude mice.

[0032] In summary, this immunodeficient nude mouse culture method, through the four-dimensional morphological characteristics of "more dense and long straight vs fewer sparse and short curved" (number, density, length, and curvature), enables non-invasive and rapid screening of newborns, breaking through the limitations of traditional genetic testing that relies on laboratory conditions. It also significantly shortens the time required to wait for the suckling mice to grow their dorsal hair and begin to appear, making identification more efficient and effective. By leveraging the high lactation performance of ICR female mice to compensate for the maternal defects of nude mice, combined with odor adaptation technology to address rejection reactions and reduce stress, the success rate of heterozygous female mice for breeding has increased from 70% with traditional methods to 95%, ensuring the continuity of homozygous nude mouse production. Furthermore, the early separation of heterozygous female mice from the same cage ensures the healthy and efficient development and growth of homozygous nude mice, improving survival rates and ensuring a continuous market supply while reducing mortality and ensuring animal welfare.

[0033] It should be noted that the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprises a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0034] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for culturing immunodeficient nude mice, characterized in that: It includes an early genotype identification step based on the morphological characteristics of the whiskers of newborn mice. Specifically, within 12 hours after the birth of the mouse, observe its whiskers on the chin and around the nose. If the whiskers are less than 10 in number, less than 0.5 mm in length, curled, and the hair follicles are sparsely distributed, it is determined to be homozygous (nu / nu); if the whiskers are greater than 20 in number, ≥1.2 mm in length, grow upright, and are closely arranged, it is determined to be heterozygous (nu / +).

2. The method for culturing immunodeficient nude mice according to claim 1, characterized in that: The treatment of heterozygotes after the identification step is: euthanasia and elimination of heterozygotes within 24 hours of birth, or reservation of heterozygous female mice according to the breeding standards.

3. The method for culturing immunodeficient nude mice according to claim 2, characterized in that: The reserved heterozygous female mice were given intensive milk replacement management, including: taking tail tip tissue for PCR genotyping identification during the weaning period (21 days of age) with primers F: 5'-GTGCTGCTGCTGCTGATG-3' and R: 5'-CTGCTGCTGCTGCTGCTA-3', and a 450bp band was amplified in the heterozygotes.

4. The method for culturing immunodeficient nude mice according to claim 3, characterized in that: The surrogate adaptation operation includes: transferring 3-day-old, 2-3g-weight heterozygous female mice to the surrogate mother cage. The surrogate mother is a heterozygous female mouse or ICR female mouse of the same species that gave birth at the same time. The pup cage is covered with surrogate mother bedding 24 hours in advance to contaminate it with the scent. Each cage of surrogate mother is paired with no more than 3 pups.

5. The method for culturing immunodeficient nude mice according to claim 4, characterized in that: During the milk replacement period, 1% whey protein powder was added to the feed to ensure that the average daily weight gain of the young mice was ≥0.3g, and the weight reached 9-12g at 3 weeks of age.

6. The method for culturing immunodeficient nude mice according to claim 1, characterized in that: The lactation management of homozygous puppies is: keep 3-4 homozygous puppies in each litter to reduce the lactation pressure of the mother mouse.

7. The method for culturing immunodeficient nude mice according to claim 1, characterized in that: It also includes health monitoring steps: establishing a whisker morphology file for suckling mice, combined with weekly blood routine monitoring, among which the homozygotes focus on monitoring the lymphocyte ratio, requiring CD3+T cells to be <1%, and constructing a "phenotype-genotype-immune indicator" triple monitoring system.

8. The method for culturing immunodeficient nude mice according to claim 1, characterized in that: The beard morphology observation is performed under a light source magnifying glass to clearly identify the number, length, curvature and hair follicle distribution of the beard.

9. The method for culturing immunodeficient nude mice according to claim 4, characterized in that: The selection criteria for the surrogate mothers were individuals with high milk production and strong maternal instincts.

10. The method for culturing immunodeficient nude mice according to any one of claims 1 to 9, characterized in that: The immunodeficient nude mice were BALB / c background nude mice.

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