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    25 October 2023, Volume 43 Issue 5
    Frontier Reviews
    Implications on the Development of Animal Disease Models from FDA Modernization Act 2.0
    Yinghan WAN, Yexin GU, Yunong YUAN, Min TANG, Li LU
    2023, 43(5):  472-481.  DOI: 10.12300/j.issn.1674-5817.2023.083
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    Laboratory animals are the foundational conditions and indispensable technical support in life science research and biomedical industry development. The scientific development of animal models of diseases is of great significance to biomedical research and industrial development. In light of the booming development of multiple emerging in vitro modelling technologies over the past decade, in 2022, the U.S. Senate unanimously passed the bill FDA Modernization Act 2.0. This bill rescinded the requirement for animal testing in investigating the safety and effectiveness of a drug—a federal mandate since 1938, and highlighted the potential of various invitro disease modeling approaches in future biomedical fields. This paper provides a comprehensive review of the latest advances and applications of in vitro disease modeling approaches in academia and industry followed by an interpretation of the FDA bill, namely cell culture, organoid, organ-on-a-chip, 3D bio-printing model and computer-based model. The paper next introduces the crossed applications of various disease models and discusses the advantages and disadvantages of each system, thereby providing insights into future trends in the use of animal disease models in China.

    Research Progress Report on Microtus fortis as a New Resource of Laboratory Animal
    Jianyun XIE
    2023, 43(5):  482-491.  DOI: 10.12300/j.issn.1674-5817.2023.114
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    Microtus fortis (reed vole) is the only mammal known to have natural resistance to Schistosomiasis japonica. Originating from schistosomiasis endemic and non-endemic areas, as well as laboratory bred voles have the same resistance to Schistosoma japonicum. After more than 30 years of laboratory cultivation of wild reed vole, a series of progress have been made in laboratory animalization. A detailed study was conducted on biological traits including growth and development, reproductive physiology, serum biochemistry, hematological indicators and tissue anatomy. At the same time, the anti-schistosomiasis characteristics and anti-schistosomiasis mechanisms of Microtus fortis were studied. The closed Dongtinghu population of Microtus fortis (S: DTMF) cultivated by Shanghai Laboratory Animal Research Center was recognized as a Chinese laboratory animal resource by the Experimental Animal Resources and Evaluation Working Committee of the Chinese Association for Laboratory Animal Sciences in 2021. This review focuses on summarizing the research progress in the biological characteristics, standardization research, genome and anti-schistosomiasis mechanism of reed vole in the past decade, especially in the implementation of the key project in the National Science and Technology Pillar Program.

    Evolution and Prospects of Laboratory Animal Management: A Case Study of Shanghai's Development in the Past Decade
    Yong ZHAO
    2023, 43(5):  492-503.  DOI: 10.12300/j.issn.1674-5817.2023.134
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    There are differences in historical and cultural beliefs, development history, and levels of technological development among different countries and regions around the world. However, they have all established corresponding laboratory animal management systems that are suitable for their national conditions. In 2001, the Ministry of Science and Technology, together with six other ministries, jointly issued the administrative licensing system for experimental animals, which was an innovative measure in China's specialized management system for experimental animals.The State Administration for Market Regulation and the National Standards Committee, based on the welfare of experimental animals and the needs of scientific research, have formulated a series of national standards for laboratory animals, and the local experimental animal management institutions, experimental animal quality testing unit and professional training base have also been established, which provide a strong guarantee for the rapid and healthy development of experimental animal science. This paper reviews the development of experimental animal management in Shanghai in the past ten years, reflects the evolution of national experimental animal management in recent years, points out the weak links in the development process, and puts forward suggestions for the innovation and development of experimental animal work.

    Progress in Establishment and Application of Laboratory Animal Models Related to Development of Male Infertility Drugs
    Shuwu XIE, Ruling SHEN, Jinxing LIN, Chun FAN
    2023, 43(5):  504-511.  DOI: 10.12300/j.issn.1674-5817.2023.120
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    As the incidence of male infertility has been increasing during recent years, it is urgent to reveal the pathogenesis of male infertility, as well as to develop the new drugs for treatment of male infertility, in order to solve the declining birth rate and aging problems. The construction and application of male infertile animal models is critical for drug development, which plays an important role in accurately evaluating the efficacy and mechanism of infertility treatment. A suitable infertility model not only can reduce the repeated drug efficacy evaluations, reduce animal usage and the cost of new drug development, but also has important reference value for subsequent clinical trial research. Male infertility laboratory animal models can be constructed through chemical, physical, endocrine, environmental estrogen, gene modification, and immune methods. This article mainly introduces the existing male infertility animal models available for drug development, and briefly introduces the application progress of each model to provide reference for the male infertility drug researchers.

    Progress in the Application of Animal Disease Models in the Medical Research on Colorectal Cancer
    Yanjuan CHEN, Ruling SHEN
    2023, 43(5):  512-523.  DOI: 10.12300/j.issn.1674-5817.2023.076
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    Colorectal cancer (CRC) is the third most common malignant tumor in the world. The latest statistics show that CRC accounts for 10% of all cancer cases worldwide and is the second leading cause of cancer deaths. CRC is a highly heterogeneous disease, the development of which is driven by functional abnormalities or epigenetic changes caused by multiple gene expression mutations, and there are different pathways that lead to tumor formation. Complex factors such as genetics, environment, ethics, and individual differences of patients themselves limit the study of CRC in humans, so the disease animal models have become an indispensable tool for the study of CRC, and play an important role in prevention, treatment, preclinical research and basic research. There are various types of CRC animal models, of which mouse models are the most widely used. According to different model establishing methods, the models are divided into spontaneous, chemically induced, transplanted tumor and genetic-engineering mouse models. Different models have different characteristics and application prospects. In this study, we focus on these mouse models of CRC in detail, and introduce the latest research progress of CRC models in rats, experimental pigs and zebrafish, to provide reference for the selection and application of animal models of CRC.

    Current Status and Reflection on the Study of Welfare for Laboratory Fish
    Liping FENG, Qi ZHU, Jinxing LIN
    2023, 43(5):  524-530.  DOI: 10.12300/j.issn.1674-5817.2023.069
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    In recent years, with the rapid development of life sciences, the use of laboratory fish in toxicology, genetics, developmental biology and medicine has increased dramatically, and they have gradually become important new model organisms. At the same time, the welfare of laboratory fish has also received increasing attention. Although the research level of experimental fish welfare is still in a relatively early stage compared to terrestrial experimental animals, developed regions such as Europe and America have established corresponding legal frameworks to safeguard the welfare of laboratory fish in research. This article elucidates the current developmental status of laboratory fish welfare, discusses the rationale behind the imperative to prioritize and enhance their welfare, deeply investigates factors influencing their welfare from the feeding stage and experimental stage. Moreover, it explores strategies for augmenting welfare standards, with the overarching aim of propelling the continual improvement of laboratory fish welfare in our country.

    Research Reports
    Fine Structure of the Trunk Kidney and Distribution of Its Secreted Exosomes in the Adult Zebrafish
    Jinxing LIN, Xindong WANG, Xuebing BAI, Liping FENG, Shuwu XIE, Qiusheng CHEN
    2023, 43(5):  531-540.  DOI: 10.12300/j.issn.1674-5817.2023.070
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    Objective To observe the fine structure of the trunk kidney in zebrafish, and to identify its secreted exosomes. Methods The microstructure and ultrastructure of the trunk kidney in zebrafish were observed by light microscopy and electron microscopy, and the particle size of exosomes was detected by nanoparticle tracking analysis (NTA). Results The trunk kidney was close and parallel to the spine in adult zebrafish. The nephron consisted of renal tubules and renal corpuscles. The renal tubules could be further divided into three types: proximal convoluted tubules, distal convoluted tubules, and cervical segments. The renal corpuscles were composed of glomerulus and renal capsules. The periodic acid-Schiff (PAS) staining results revealed that there were abundant glycogen granules in the proximal convoluted tubules, with brush-like outline in the apical surface of epithelial cells. Under transmission electron microscopy (TEM), there were exosomes distributed in the lumen of renal tubules, with numerous late endosomes and few number of multivesicular bodies (MVBs) in the cytoplasm of the epithelial cells concentrating on the apical side. Meanwhile, MVBs were also distributed in the apical regions of the renal tubules and the podocytes of the renal glomeruli. Immunohistochemical staining results showed that CD9, CD63 and TSG101 were strongly expressed in the lumen surface of the renal tubules, but weakly expressed in the corpuscles and lumen. NTA and TEM results showed that the exosomes isolated from zebrafish trunk kidney were saucer-like outline, and the particle size mode was 144.4 nm, which was consistent with the characteristics of morphological futures of exosome. Conclusion The zebrafish somatic kidney has the typical structure of the mammalian kidney and is the urinary organ in the body. The renal tubules have the ability to secrete exosomes, and their formation is a process of releasing poly-vesicles to the free surface of epithelial cells into the extracellular space. This study laid a morphological foundation for further study of exosomes in urinary function in aquatic experimental animals as well as the development and application of related models.

    Generation of 12 Drosophila Transgenic Negative Control Lines Based on Site-specific ΦC31 Integrase and pUASTattB Vector
    Longmei XU, Ruling SHEN, Chun FAN, Wei WU
    2023, 43(5):  541-547.  DOI: 10.12300/j.issn.1674-5817.2023.100
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    Objective Construction of a negative control line for the Drosophila transgenic system based on ΦC31 integrase and vector plasmid pUASTattB to provide a more scientific negative control for transgenic Drosophila research experiments. Methods The vector plasmid pUASTattB was microinjected into four different genetic backgrounds Drosophila lines attP-25C6, attP-68A4, attP-75B1 and attP-86F8 embryos carrying ΦC31 integrase. All of the injected embryos were incubuated to get G0 adults, and each of them was crossed with balancer stock ywR13S separately in a single vial (1 adult of the G0 generation and 3 of the ywR13S in each vial). The probability of successful insertion was calculated by observing the colour of the compound eyes of the G1 generation of Drosophila to determine whether there was a mini-White insertion. The G1 generation Drosophila adults successfully inserted into mini-White were then selected to make single-vial crosses (one G1 generation male Drosophila crossed with three virgins of balancer Drosophila line) with each of the three balancer Drosophila strains DB, ywR13S and yw122, respectively, for balanced seed preservation. The genomic DNA of the conserved Drosophila lines was extracted and the vector plasmid pUASTattB was identified for transfer by PCR. Results 12 Drosophila strains were obtained, all of which were red-eyedDrosophila melanogaster carrying the mini-White marker, and were identified by PCR as having the pUASTattB sequence insertion. Conclusion The 12 transgenic Drosophila strains can meet the negative control requirements for the transgenic fly research experiments that constructed with pUASTattB as the vector basically, enriching the Drosophila resources in the National Drosophila Resource Center of China.

    Establishing a Genetic Detection Protocol of Single Nucleotide Polymorphisms Panels in Inbred Rats Based on Multiplex PCR-LDR
    Liya ZHAO, Liju NI, Caiqin ZHANG, Jianping TANG, Yangzheng YAO, Yanyan NIE, Xiaoxue GU, Ying ZHAO
    2023, 43(5):  548-558.  DOI: 10.12300/j.issn.1674-5817.2023.073
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    Objective To establish a set of single nucleotide polymorphisms (SNP) detection protocol for inbred rats based on multiplex PCR-ligase detection reaction (LDR). Methods A total of 40 rats SNP sites were selected on chromosomes 1-20 and X of rats among 5 inbred strains of rats, and the 40 SNP sites were randomly divided into four groups. A genetic detection protocol for 4 groups of SNP in inbred rats based on multiplex PCR-LDR technology was constructed. 9 commonly used rat strains from two other domestic rat suppliers were detected by this protocol. Finally, the feasibility of this protocol was verified by comparing the amplification effects of different DNA polymerases by a third-party laboratory. Results When using the constructed SNP detection protocol for inbred rats to test 5 rat strains, all sites in each sample obtained good amplification results. The 9 commonly used rat strains from two other rat suppliers in china were also well amplified by this SNP detection protocol, and 40 SNPs were homozygous in each Inbred strain. The results of detection of the same rat DNA samples with three different DNA polymerases showed that the Multiplex PCR Kit, AmpliTaq Gold 360 DNA polymerase and Platinum II Taq hot start DNA polymerase had electrophoretic peaks of amplification products at all SNP sites in groups 1 to 3, and Platinum II Taq hot start DNA polymerase had one less electrophoretic peak of the amplification products at the SNP sites in group 4. In addition, inter-laboratory comparisons showed consistent results for the same amplification system. Conclusion Based on multiplex PCR-LDR technology, this study successfully established a SNP detection protocol for rats covering all autosomes and X chromosomes with the excellent stability and repeatability.

    Investigation Report on the Production and Utilization Status of Experimental Mini-pigs
    Shuzhen ZHANG, Yanguang ZHAO
    2023, 43(5):  559-565.  DOI: 10.12300/j.issn.1674-5817.2023.075
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    Objective To understand the current situation of the experimental miniature pig industry and promote its good development. Methods Using Questionnaire Star to design the survey content, the survey was conducted by targeted push and voluntary filling. The valid questionnaires collected were classified and summarized by EXCEL2010 software. Results A total of 35 entities participated in the survey, including 12 production entities and 23 user entities. There were 1 623 employees in the 35 entities, 927 (57.11%) with college degree or below, and 696 (42.89%) with bachelor degree or above. The largest number of employees have majors in animal science and animal medicine. The salary level of employees in the production entity is higher than that of the user entity, but the research output (published papers and patents) rate of the user entity in the past 5 years is higher than that of the production entity. The current inventory of miniature pigs in 12 production entities was 5 353, of which 3 471 (64.84%) were Bama pigs and 5 243 (97.95%) were ordinary grade pigs. The vaccination rate of swine fever and foot-and-mouth disease was 100%, and the animal mortality rate of seven entities was ≤3.0%. The profit of ordinary grade miniature pigs was 954 yuan/head, and that of SPF grade miniature pigs was 10 037 yuan/head. Conclusion It is suggested to strengthen market supervision, expand the channels for introducing professional and technical talents, accelerate the research of new quality testing technologies, promote the rapid transformation of miniature pig scientific research achievements, and establish a public service platform to strengthen information communication between supply and demand sides. We hope to work together with our colleagues to jointly promote the normalized, standardized, healthy and stable development of the experimental miniature pig industry.

    Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice
    Lingzhi YU, Jianyun XIE, Liping FENG, Xiaofeng WEI
    2023, 43(5):  566-573.  DOI: 10.12300/j.issn.1674-5817.2023.022
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    Objective To establish a method for rapid and sensitive detection of Staphylococcus aureus. Methods The specific gene nuc of Staphylococcus aureus was selected as the target gene. A pair of specific primers and a TaqMan probe were designed and synthesized according to the published sequence of the nuc gene. Establish a nucleic acid detection method for nuc gene using fluorescence quantitative PCR technology, and apply it clinically in the detection of fecal samples from rats and mice. Results The DNA extracted from Staphylococcus aureus and other non-Staphylococcus aureus strains was detected by qPCR. The results showed that Staphylococcus aureus had a specific amplification curve, while other non-Staphylococcus aureus did not, indicating that the designed primers and probes were specific for Staphylococcus aureus. The sensitivity of this method was determined by diluting the DNA of Staphylococcus aureus by 10 times. The results showed that the detection limit of this method was 10 fg DNA, which was 2 orders of magnitude higher than that of ordinary PCR method. A total of 91 clinical samples were detected in this study, of which 4 rat samples from the same facility had a typical S-curve. The PCR products were sequenced and BLAST compared. The gene sequence of this sample was 100% similar to that of Staphylococcus aureus, indicating that the sample was positive for the nucleic acid of Staphylococcus aureusnuc gene, with a positive rate of 4.40%. The result was consistent with that obtained by bacterial culture method. The nucleic acid extraction adopted a full-automatic nucleic acid purification instrument, and the time required from nucleic acid extraction to detection result determination was less than 1.5 h. Conclusion The qPCR method established in this study to identify Staphylococcus aureus with nuc gene as the target gene has the advantages of fast, high sensitivity and specificity, and can be used for the detection of Staphylococcus aureus in feces of rats and mice.

    Application of Optimized Latex Perfusion Technique in the Establishment of Craniofacial Venous Model in Mice
    Chengji WANG, Jue WANG, Haijie WANG, Weisheng LU, Yan SHI, Zhengye GU, Mingqiu WAN, Ruling SHEN
    2023, 43(5):  574-578.  DOI: 10.12300/j.issn.1674-5817.2023.095
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    Objective Optimize the latex perfusion technique and apply it to the construction of a murine craniofacial venous vascular model. Methods A total of nine 8-week-old male C57BL/6 mice weighing (25.0±1.3) g were randomly divided into three groups: 60% latex physiological saline group, 60% latex heparin group, and 30% latex heparin group. After completion of the perfusion, the specimens were immersed in 4 °C formalin fixative for 24 h, followed by dissection, observation, and measurement of the extracranial blood vessel diameters. Results After 200 μL latex perfusion solution was injected into the external jugular vein, the supraorbital vein, infraorbital vein, temporal vein, retrofacial vein, masseter vein and external jugular vein were perfused in each group.After comparing the perfusion degree of the distal branches of blood vessels, sublingual vein and tip venule, it was found that the 30% latex heparin group had the best perfusion effect, followed by the 60% latex heparin group, and the 60% latex saline group had the worst perfusion effect. Conclusion The optimized latex perfusion technique can effectively infuse the veins in the head and face of mice, and this technique can provide a good reference for the study of the direction and morphology of facial veins in mice.