Of mice and microflora: considerations for genetically engineered mice

The phenotype of genetically engineered mice is a combination of both genetic and environmental factors that include the microflora of the mouse. The impact a particular microbe has on a mouse reflects the host-microbe interaction within the context of the mouse genotype and environment. Although often considered a confounding variable, many host-microbe interactions have resulted in the generation of novel model systems and characterization of new microbial agents. Microbes associated with overt disease in mice have been the historical focus of the laboratory animal medical and pathology community and literature. The advent of genetic engineering and the complex of mouse models have revealed previously unknown or disregarded agents that now oblige the attention of the biomedical research community. The purpose of this article is to describe and illustrate how phenotypes can be affected by microflora by focusing on the infectious diseases present in genetically engineered mouse (GEM) colonies of our collective institutions and by reviewing important agents that are rarely seen in most research facilities today. The goal is to introduce the concept of the role of microflora on phenotypes and in translational research using GEM models.     

Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice

Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.     

Pathobiology of aging mice and GEM: background strains and experimental design

The use of induced and spontaneous mutant mice and genetically engineered mice (and combinations thereof) to study cancers and other aging phenotypes to advance improved functional human life spans will involve studies of aging mice. Genetic background contributes to pathology phenotypes and to causes of death as well as to longevity. Increased recognition of expected phenotypes, experimental variables that influence phenotypes and research outcomes, and experimental design options and rationales can maximize the utility of genetically engineered mice (GEM) models to translational research on aging. This review aims to provide resources to enhance the design and practice of chronic and longevity studies involving GEM. C57BL6, 129, and FVB/N strains are emphasized because of their widespread use in the generation of knockout, transgenic, and conditional mutant GEM. Resources are included also for pathology of other inbred strain families, including A, AKR, BALB/c, C3H, C57L, C58, CBA, DBA, GR, NOD.scid, SAMP, and SJL/J, and non-inbred mice, including 4WC, AB6F1, Ames dwarf, B6, 129, B6C3F1, BALB/c,129, Het3, nude, SENCAR, and several Swiss stocks. Experimental strategies for long-term cross-sectional and longitudinal studies to assess causes of or contributors to death, disease burden, spectrum of pathology phenotypes, longevity, and functional healthy life spans (health spans) are compared and discussed.     

一种犬细小病毒基因工程抗体的制备

本研究旨在利用HEK-293细胞系制备鼠源犬细小病毒（canine parovirus，CPV）基因工程抗体并检测其生物活性。通过抗体亚型检测试剂盒检测CPV单克隆抗体亚型；采用间接ELISA检测CPV单克隆抗体的亲和力和特异性；经RACE-PCR获得CPV单克隆抗体的可变区序列，将可变区序列与鼠源抗体恒定区序列连接；分别构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H，将载体共转染HEK-293细胞，采用血凝抑制与中和试验的方法检测鼠源CPV基因工程抗体生物活性；采用HEK-293F细胞悬浮表达并用间接ELISA方法检测鼠源CPV基因工程抗体的表达量；用Protein A亲和层析柱纯化鼠源CPV基因工程抗体后进行SDS-PAGE鉴定；间接免疫荧光检测纯化后鼠源CPV基因工程抗体的活性。结果显示，CPV单克隆抗体亚型为IgG_2b，亲和力常数6个Ka平均值为1.02×10¹¹ L/mol，只与CPV VLPs发生反应。琼脂糖凝胶电泳结果显示，试验成功构建真核表达载体pcDNA3.1（+）-L和pcDNA3.1（+）-H；HEK-293和HEK-293F细胞培养上清液血抑效价分别为1∶2⁴和1∶2⁶，中和试验结果显示，HEK-293和HEK-293F细胞培养上清液中和效价分别为1∶152和1∶1 290；鼠源CPV基因工程抗体在HEK-293F细胞中的表达量为5.97 mg/L，SDS-PAGE分析在55和25 ku处出现条带，表明鼠源CPV基因工程抗体成功在HEK-293F细胞中表达并纯化。间接免疫荧光检测结果表明，纯化后鼠源CPV基因工程抗体具有良好的生物活性。本研究在HEK-293F细胞中成功表达具有中和活性、纯度较高的鼠源CPV基因工程抗体，为今后CPV基因工程抗体药物的研发奠定基础。     

Genetically engineered animal models for Marfan syndrome: challenges associated with the generation of pig models for diseases caused by haploinsufficiency

Recent developments in reproductive biology have enabled the generation of genetically engineered pigs as models for inherited human diseases. Although a variety of such models for monogenic diseases are currently available, reproduction of human diseases caused by haploinsufficiency remains a major challenge. The present study compares the phenotypes of mouse and pig models of Marfan syndrome (MFS), with a special focus on the expressivity and penetrance of associated symptoms. Furthermore, investigation of the gene regulation mechanisms associated with haploinsufficiency will be of immense utility in developing faithful MFS pig models.     

The gene or not the gene--that is the question: understanding the genetically engineered mouse phenotype

Embryonic stem cells have had a significant impact on understanding gene function and gene interactions through the use of genetically engineered mice. However, the genetic context (ie, mouse strain) in which these modifications in alleles are made may have a considerable effect on the phenotypic changes identified in these mice. In addition, tissue- and time-specific gene expression systems may generate unanticipated outcomes. This article discusses the history of embryonic stem cells, reviews how mouse strain can affect phenotype (using specific examples), and examines some of the caveats of conditional gene expression systems.     

Pathology methods for the evaluation of embryonic and perinatal developmental defects and lethality in genetically engineered mice

The normal embryonic development of organs and other tissues in mice and all species is preprogrammed by genes. Inactivation of a gene involved in any stage of normal embryonic development can have severe consequences leading to embryonic or postnatal developmental defects and lethality. Pathology methods are reviewed for evaluating normal and abnormal placenta and embryo, especially after E12.5. These methods include pathology protocols for necropsy and histopathology in addition to references that will provide additional knowledge for embryo assessment including histology atlases and advanced embryo imaging techniques.     

Genetic alterations in thyroid cancer: the role of mouse models

Thyroid carcinomas are the most common endocrine neoplasms in humans, with a globally increasing incidence. Thyroid follicular cells and neuroendocrine (parafollicular) C cells are each susceptible to neoplastic transformation, resulting in thyroid cancers of differing phenotypes with unique associated genetic mutations and clinical outcomes. Over the past 15 years, several sophisticated genetically engineered mouse models of thyroid cancer have been created to further our understanding of the genetic events leading to thyroid carcinogenesis in vivo. The most significant mouse models of papillary, follicular, anaplastic, and medullary thyroid carcinoma are highlighted, with particular emphasis on the relationship between the relevant oncogenes in these models and genetic events in the naturally occurring human disease. Limitations of each model are presented, and the need for additional models to better recapitulate certain aspects of the human disease is discussed.     

Current status of foot-and-mouth disease vaccines including the use of genetic engineering

SUMMARY Foot-and-mouth disease virus (FMDV) vaccines are used to protect animals against infection by the 7 FMDV serotypes composed of greater than 60 FMDV subtypes. Because of problems of both live attenuated and inactivated FMDV vaccines and also because of the very large market for an effective safe vaccine, research into other types of vaccines has been undertaken. One of the 4 virus structural proteins, VP1, is believed to be the main protein that stimulates virus neutralising antibodies and studies have concentrated on its potential as a subunit vaccine. Genetic engineering has been used to clone the VF1 gene of FMDV and VP1 synthesised from the cloned gene has been used in experimental vaccine studies. The studies in small numbers of cattle and pigs demonstrated that 2 vaccinations with genetically engineered VP1 could confer protection against FMDV challenge. However, there are a number of areas that need further research before such a genetically engineered vaccine could be used commercially. The use of chemically synthesised antigenic fragments of VP1 has recently been reported, and these synthetic fragments appear to be potentially better at producing immunity to FMDV than the whole genetically engineered VP1 protein, perhaps because of conformational problems in the presentation of whole VP1. Other possible future directions in the research and in the development of safe, effective FMDV vaccines are discussed. In conclusion, although very significant progress has been made in cloning FMDV-VP1 genes, we are still far from a genetically engineered VP1-FMDV subunit vaccine. In the meantime, properly inactivated and safety-tested FMDV vaccines will continue to be used and to be of benefit to the livestock industry in countries where foot-and-mouth is endemic or in combating introductions of the disease.     

基因工程药物研究概况

对新发展起来的产业基因工程药物的研究作了简要的概述,通过对其发展历史及当前的几种药物的叙述,预测其发展前景。     

3-dimensional imaging modalities for phenotyping genetically engineered mice

A variety of 3-dimensional (3D) digital imaging modalities are available for whole-body assessment of genetically engineered mice: magnetic resonance microscopy (MRM), X-ray microcomputed tomography (microCT), optical projection tomography (OPT), episcopic and cryoimaging, and ultrasound biomicroscopy (UBM). Embryo and adult mouse phenotyping can be accomplished at microscopy or near microscopy spatial resolutions using these modalities. MRM and microCT are particularly well-suited for evaluating structural information at the organ level, whereas episcopic and OPT imaging provide structural and functional information from molecular fluorescence imaging at the cellular level. UBM can be used to monitor embryonic development longitudinally in utero. Specimens are not significantly altered during preparation, and structures can be viewed in their native orientations. Technologies for rapid automated data acquisition and high-throughput phenotyping have been developed and continually improve as this exciting field evolves.     

Neurotoxic effects of ivermectin administration in genetically engineered mice with targeted insertion of the mutated canine ABCB1 gene

Objective-To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies. Animals-14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted. Procedures-Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5). Each was observed for clinical signs of toxic effects from 0 to 7 hours following drug administration. Results-After ivermectin administration, considerable differences were observed in drug sensitivity between the 2 types of mice. The genetically engineered mice with the mutated canine ABCB1 gene had signs of severe sensitivity to ivermectin, characterized by progressive lethargy, ataxia, and tremors, whereas the wild-type control mice developed no remarkable effects related to the ivermectin. Conclusions and Clinical Relevance-The ivermectin sensitivity modeled in the transgenic mice closely resembled the lethargy, stupor, disorientation, and loss of coordination observed in ivermectin-sensitive Collies with the ABCB1-1Δ mutation. As such, the model has the potential to facilitate toxicity assessments of certain drugs for dogs that are P-glycoprotein substrates, and it may serve to reduce the use of dogs in avermectin derivative safety studies that are part of the new animal drug approval process.     

Non‐phytate phosphorus requirements and efficacy of a genetically engineered yeast phytase in male lingnan yellow broilers from 1 to 21 days of age

This experiment was conducted to investigate the requirement of non‐phytate phosphorus (nPP) and efficacy of a genetically engineered yeast phytase in performance and tibia characteristics by male Lingnan Yellow broilers from 1 to 21 days of age. A total of 2640 1‐day‐old male chicks were randomly allotted to one of 11 dietary treatments, which consisted of six replicate floor pens with 40 birds per pen. All treatments had the same levels of all nutrients except for phosphorus and phytase. The control group (treatment 1) was fed the basal diet without dicalcium phosphate or phytase supplementation. Dietary concentrations of nPP were 0.11%, 0.19%, 0.27%, 0.35%, 0.43%, 0.51% and 0.59% respectively for treatments 1, 2, 3, 4, 5, 6 and 7, through addition of dicalcium phosphate (chemistry grade) to the basal diet. Diets 8–11 were supplemented with a genetically engineered yeast phytase 250, 500, 750 U/kg and a commercial phytase product 500 U/kg in basal diet respectively. The results showed that 0.46% and 0.51% nPP were required for maximum body‐weight gain and optimum tibia development indicators respectively. However, 0.59% nPP had a negative effect on bird growth. The equivalency value of the genetically engineered yeast phytase was estimated to be 874 U/kg to liberate 0.1% nPP.     

Generation of RUNX3 knockout pigs using CRISPR/Cas9‐mediated gene targeting

Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies.     

Hermaphroditism in 3 chimeric mice

Hermaphroditism was diagnosed in three, 6-month-old, male, chimeric mice generated by microinjection of 129/Ola XY recombinant embryonic stem cells into unsexed C57BL/6 blastocysts. Grossly, mice Nos. 1 and 2 had perigenital masses and hydrometra. All mice had unilateral ovaries and cystic endometrial hyperplasia. Mice Nos. 1 and 3 also had contralateral testes and epididymides. Histologically, mice Nos. 1 and 3 were true hermaphrodites with unilateral ovotestes, while mouse No. 2 was a pseudohermaphrodite with ovarian tissue only. The presence of a uterus with cystic endometrial hyperplasia in these mice resembles XY pseudohermaphroditism in miniature schnauzers. The mice were determined to be 95 to 100% chimeric via haircoat color; however, the presence of both male and female sex organs in these phenotypically male mice suggests otherwise. Published reports note incidences for sex chimeras and hermaphroditism in genetically engineered mice of 50% and 20%, respectively. Hermaphroditism is expected to increase as the numbers of chimeric mice rise with technical advances in genetic engineering.     

Development of an Efficient Method for Genotyping at the gd Locus in NC/Sgn Mice Based on PCR-RFLP Analysis

Growth deficit (gd) is a recessive mutation that occurs spontaneously in the inbred NC/Sgn mouse strain. Because homozygotes (gd/gd) of both sexes are sterile, they must be produced by mating putative heterozygous carriers (+/gd) whose phenotypes are essentially the same as those of wild-type +/+ mice. The objectives of this study were to develop an efficient method that distinguished a gd allele from a wild-type allele and, if possible, to identify nucleotide substitutions responsible for the gd mutation. The location of the gd locus was estimated to be at 58.3 Mbp on chromosome 4, over which Musk is located. An A-to-G base substitution, which resulted in an M826V amino acid exchange, was identified within a tyrosine kinase domain of Musk. This base substitution disrupted a recognition site for NlaIII; this allowed for discriminating the gd allele from the wild-type allele using PCR-RFLP analysis. When 130 (C57BL/6J × NC/Sgn-gd) F(2) mice were genotyped by PCR-RFLP analysis, all 32 growth-retarded F(2) mice were judged to have the gd/gd genotype. Musk mutations are known to cause congenital myasthenia, which is accompanied by growth retardation, postnatal lethality, and development of a hunchback. These were the typical phenotypes of gd/gd mutants. Although we cannot rule out the possibility that the neighboring genes around the Musk locus are related to the gd phenotype, gd could possibly be classified as a mutant allele of Musk.     

Phenotypic plasticity induced using high ambient temperature during embryogenesis in domesticated zebrafish,Danio rerio

Ambient temperature during early stages of life has a substantial effect on physiological processes, eliciting phenotypic plasticity during zebrafish developmental stages. Zebrafish are known to possess a noteworthy ability to modify their phenotype in dependence of environmental factors. However, there is a poor understanding of the effects of temperature during embryogenesis, which influences the biological functions such as survival ability and masculinization in later developmental stages. Since the middle embryonic phase (pharyngula period) is genetically the most conserved stage in embryogenesis, it is very susceptible to embryonic lethality in developmental processes of vertebrates. Here, we tested the effect of transient perturbations (heat shock) during early development (5–24 hr post‐fertilization; hpf) at 35°C compared to control group at 28°C, on survival ability of zebrafish to study the embryonic and post‐embryonic mortality. We studied the variation of heat‐induced masculinization among and across the families in response to high temperature. Furthermore, morphometric traits of adult zebrafish at different developmental time points were measured in order to estimate the temperature × sex interaction effect. We found the highest embryonic mortality around the gastrula and segmentation periods in both experimental groups, with significantly lower survival ability in the temperature‐treated group (73.30% ± 0.58% vs. 70.19% ± 0.57%, respectively). A higher hatching success was observed in the control group (71.08% ± 0.61%) compared to the heat‐induced group (67.95% ± 0.60%). A distinct reduction in survival ability was also observed in both experimental groups during the first two weeks after hatching, followed by a reduced level of changes thereafter. We found sex ratio imbalances across all families, with 25.2% more males under temperature treatment. Our study on growth performance has shown a positive effect of increased temperature on growth plasticity, with a greater impact on female fish in response to high ambient temperature.     

富含蛋氨酸醇溶蛋白在产朊假丝酵母中的表达研究

为构建表达富含蛋氨酸醇溶蛋白的产朊假丝酵母工程菌株,利用基因工程技术,将玉米δ—10kD-醇溶蛋白基因（zein）,酵母的GAP启动子（GAP—P）、终止子（GAP—t）拼接合成表达框,以18S rDNA片段为整合介导区．用放线菌酮（CYH）抗性基因作为筛选标记,结合pBR322质粒构建了同源整合表达载体．把zein基因同源重组整合到产朊假丝酵母染色体上,构建表达zein基因的产朊假丝酵母工程菌株。蛋氨酸含量测定结果显示,重组菌株的蛋氨酸表达量比起始菌株提高了17．14％。该产朊假丝酵母工程菌的构建为下一步提高其蛋氨酸产量的研究和应用打下了良好的基础。     

Internet and print resources to facilitate pathology analysis when phenotyping genetically engineered rodents

Genetically engineered mice and rats are increasingly used as models for exploring disease progression and mechanisms. The full spectrum of anatomic, biochemical, and functional changes that develop in novel, genetically engineered mouse and rat lines must be cataloged before predictions regarding the significance of the mutation may be extrapolated to diseases in other vertebrate species, including humans. A growing list of reference materials, including books, journal articles, and websites, has been produced in the last 2 decades to assist researchers in phenotyping newly engineered rodent lines. This compilation provides an extensive register of materials related to the pathology component of rodent phenotypic analysis. In this article, the authors annotate the resources they use most often, to allow for quick determination of their relevance to research projects.