Development of mouse embryos in vitro is affected by strain and culture medium

One-cell and two-cell embryos from three random-bred strains of mice–CF1, Dub:(ICR), and CFW (Swiss-Webster)–were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.     

Strain-Dependent Differences in the Efficiency of Transgenic Mouse Production

Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production.     

不同品系小鼠感染甲型流感病毒肺小血管内血栓形成

目的本实验旨在观察不同品系小鼠感染甲型流感病毒后肺组织内血栓形成的情况。方法使用H1N1病毒A/California/7/2009（CA7）株和H3N2病毒A/Brisbane/10/07株,对BALB/C小鼠、Scid小鼠、NOD/LTJ小鼠、BALB/C-nu小鼠、NOD-Scid小鼠和icosl-KO小鼠经乙醚麻醉后进行滴鼻攻毒。检测小鼠感染后肺组织病毒拷贝数并观察肺组织病理学改变。结果 H1N1和H3N2滴鼻攻毒的各组小鼠均染毒,病理表现为程度略有差异的间质性肺炎。13只H1N1病毒感染小鼠和6只H3N2感染小鼠在肺组织中观察到多个小血管内有血栓形成,血栓成分主要为纤维素和血小板。结论各品系小鼠感染H1N1和H3N2流感病毒后均可能出现肺组织内血栓形成。     

Mouse models of oxidative phosphorylation dysfunction and disease

Oxidative phosphorylation (OXPHOS) deficiency results in a number of human diseases, affecting at least one in 5000 of the general population. Altering the function of genes by mutations are central to our understanding their function. Prior to the development of gene targeting, this approach was limited to rare spontaneous mutations that resulted in a phenotype. Since its discovery, targeted mutagenesis of the mouse germline has proved to be a powerful approach to understand the in vivo function of genes. Gene targeting has yielded remarkable understanding of the role of several gene products in the OXPHOS system. We provide a “tool box” of mouse models with OXPHOS defects that could be used to answer diverse scientific questions.     

Quantitative analysis of retromer complex-related genes during embryo development in the mouse

The retromer complex is a heteropentameric protein unit associated with retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Functional silencing study of the Vps26a gene indicated the important role of the retromer complex during early developmental stages in the mouse. However, individual expression patterns and quantitative analysis of individual members of the retromer complex during the early developmental stages has not been investigated. In this study, we conducted quantitative expression analysis of six retromer complex genes (Vps26a, Vps26b, Vps29, Vps35, Snx1, and Snx2) and one related receptor gene (Ci-mpr) during the eleven embryonic stages with normal MEF (mouse embryonic fibroblast) and Vps26a(-/-) MEF cells. Remarkably, except for Vps26a (maternal expression pattern), all tested genes showed maternal-zygotic expression patterns. And five genes (Vps26b, Vps29, Vps35, Snx2, and Ci-mpr) showed a pattern of decreased expression in Vps26a(-/-) MEF cells by comparative analysis between normal MEF and Vps26a(-/-) MEF cells. However, the Snx1 gene showed a pattern of increased expression in Vps26a(-/-) MEF cells. From our results, we could assume that retromer complex-related genes have important roles during oocyte development. However, in the preimplantation stage, they did not have significant roles.     

Effect of centrifugation of mouse eggs on their development in vitro and in vivo

Fertilized mouse eggs were centrifuged at 5,000g, 10,000g, 15,000g, and 20,000g for 3 and 10 min or at 25,000g for 10 min. The pronuclei of centrifuged eggs became more distinctive than those of uncentrifuged eggs. The proportion of centrifuged and uncentrifuged eggs that developed to blastocysts was not significantly different. There was also no significant difference in the proportion of live fetuses obtained following the transfer of blastocysts developed from centrifuged and uncentrifuged eggs. This study demonstrated that there is no effect of centrifugation on the subsequent development of mouse eggs.     

Postnatal changes in the expression of p60c-Src in mouse testes

Src family non-receptor tyrosine kinases are involved in signaling pathways which mediate cell growth, differentiation, transformation and tissue remodeling in various organs. In an effort to elucidate functional involvement of p60c-Src (c-Src) in spermatogenesis, the postnatal changes in c-src mRNA and c-Src protein together with kinase activity and subcellular localization were examined in mouse testes. c-src mRNA levels in testes increased during the first 2 weeks of postnatal development (PND). Following a decrease at puberty (PND 28), the c-src mRNA levels re-increased at adulthood (PND 50). Src kinase activity of testes was low at PND 7 but sharply increased prepubertally (PND 15) and highest at adulthood. Upon Western blotting, the level of c-Src protein was the highest in prepubertal testes but rather decreased in adult testes at PND 50. In adult testes, ubiquitination of c-Src proteins was apparent compared with immature one at PND 7, suggesting active turnover of c-Src by ubiquitination. In immature testes, c-Src immunoreactivity was largely found in the cytoplasm of the Sertoli cells. By contrast, in pubertal and adult testes intense immunoreactivity was localized at the adluminal and basal cytoplasm of Sertoli cells bearing elongated spermatids and early germ cells, respectively. The immunoreactivity of c-Src in the Leydig cells was increased during pubertal development, suggesting the functional involvement of c-Src in differentiated adult Leydig cells. Throughout postnatal development, some spermatogonia and spermatocytes showed intensive c-Src immunoreactivity compared with other germ cells, suggesting a possible role of c-Src in germ cell death. Taken together, it is suggested that c-Src may participate in the remodeling of the seminiferous epithelia and functional differentiation of Leydig cells during the postnatal development of mouse testes.     

Functional diversities of two activity components of circadian rhythm in genetical splitting mice (CS strain)

CS mice, an inbred strain, showed two distinctive characteristics in the circadian rhythm of locomotor activity: (1) large variation in the freerunning period, and (2) spontaneous rhythm splitting under continuous darkness. In the splitting rhythm there was a positive correlation between the freerunning period of the evening component and the activity time of the morning component. The phase-shifting effect of a 15-min light pulse was examined on the two activity components of the splitting rhythm. There were significant differences in the amount of light-induced phase response between the two components. A light pulse during the late subjective night induced a phase advance shift only in the morning component, while a light pulse during the early subjective night induced a phase delay shift only in the evening component. These results indicate functional diversities of the two activity components in the circadian locomotor rhythm of CS mice, and suggest that the circadian system in CS mice consists of two mutually coupled oscillators which have different circadian periods and different responsiveness to light. The CS mouse is a useful model to explore a genetic background of oscillator coupling in the circadian system of nocturnal rodents. Accepted: 19 November 1998     

Effect of hepatocyte growth factor on long term hematopoiesis of human progenitor cells in transgenic-severe combined immunodeficiency mice

Hepatocyte growth factor (HGF), which was originally isolated as a liver generating factor, enhances hematopoiesis. To study the effect of HGF on hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we generated severe combined immunodeficiency (SCID) mice producing human (h) HGF and/or stem cell factor (SCF) by transferring the relevant genes to fertilized eggs, and then transplanted hematopoietic progenitors from human cord blood into the transgenic (Tg) SCID mice. Six months after transplantation, a significantly larger number of human cells were found in the Tg SCID mice than in non-Tg controls. Characteristically, the recipient SCID mice producing h HGF (HGF-SCID) had a significantly increased number of h CD41+ cells, whereas the SCF-SCID recipients had more CD11b+ cells. Significantly large numbers of CD34+ progenitors were found in the SCID mice transferred with both h HGF and h SCF genes (HGF/SCF-SCID) when compared with HGF-SCID or SCF-SCID mice. These results imply that HGF supports the differentiation of progenitors in megakaryocyte lineage, whereas SCF supports that in myeloid lineage. The results also imply that HGF acts on HSCs/HPCs as a synergistic proliferative factor combined with SCF. We have demonstrated the advantage of the human cytokine-producing animal in the maintenance of human HSCs.     

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium

Summary Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C₃H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Tracing location by applying Emerald luciferase in an early phase of murine endometriotic lesion formation

The pathogenesis of endometriosis has not been fully elucidated. We focused on the behavior of the ectopic endometrium, that is, the origin of the endometriotic lesion, before adhering to the peritoneal cavity. To observe lesion formation in the very early phase, we developed a novel endometriosis animal model using bioluminescence technology. We established a new transgenic mouse that expressed Emerald luciferase (ELuc) under the control of the CAG promoter. This transgenic mouse, called the CAG-ELuc mouse, showed strong bioluminescence emission; we succeeded in tracing the lesion location by the emission of ELuc. The accuracy of tracing by ELuc was high (57.7–100% of correspondence) and depended on the dosage of E2 administration. In the very early phase after transplantation, the process of lesion formation can be observed non-invasively and chronologically. We have verified that the preferred location of the uterus (transplanted grafts) was fixed immediately after the transplantation of the grafts.     

Immunologic studies on Heligmosomoides polygyrus infection in the mouse: the dynamics of single and multiple infections and the effect of DDT upon acquired resistance

Neilson J.T. McL., Forrester D.J. and Thompson N.P. 1973. Immunologic studies on Heligmosomoides polygyrus infection in the mouse: The dynamics of single and multiple infections and the effect of DDT upon acquired resistance. International Journal for Parasitology3: 371–378. Swiss Webster mice were given infections of 100,200, 300 and 400 Heligmosomoides polygyrus (= Nematospiroides dubius) larvae respectively at intervals of 4 weeks. Where appropriate, the preceding infection was terminated with anthelmintic 7 days prior to the subsequent infection. Animals were killed at regular inteivals following each infection and the worm burdens compared with those found in control mice given a primary infection of similar size. The expulsion of worms in mice given three previous infections occurred after day 3 and before day 7 postinfection indicating that those larvae moulting from the fourth to fifth stages may be most susceptible to the host's resistance mechanisms. The administration of p,p'-DDT to hyperinfected mice did not interfere with the immunologic expulsion of worms.     

A new fast real‐time PCR method for the identification of three sibling Apodemus species (A. sylvaticus,A. flavicollis,and A. alpicola) in Italy

The identification of field mice Apodemus flavicollis, Apodemus sylvaticus, and Apodemus alpicola represents a challenge for field scientists due to their highly overlapping morphological traits and habitats. Here, we propose a new fast real‐time PCR method to discriminate the three species by species‐specific TaqMan assays. Primers and probes were designed based on the alignment of 54 cyt‐b partial sequences from 25 different European countries retrieved from GenBank. TaqMan assays were then tested on 133 samples from three different areas of Italy. Real‐time PCR analysis showed 92 samples classified as A. flavicollis, 13 as A. sylvaticus, and 28 as A. alpicola. We did not observe any double amplification and DNA sequencing confirmed species assignment obtained by the TaqMan assays. The method is implementable on different matrices (ear tissues, tail, and blood). It can be used on dead specimens or on alive animals with minimally invasive sampling, and given the high sensitivity, the assay may be also suitable for degraded or low‐DNA samples. The method proved to work well to discriminate between the species analyzed. Furthermore, it gives clear results (amplified or not) and it does not require any postamplification handling of PCR product, reducing the time needed for the analyses and the risk of carryover contamination. It therefore represents a valuable tool for field ecologists, conservationists, and epidemiologists.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

博来霉素诱导G57BL/6与ICR小鼠肺间质纤维化模型的比较

目的探讨C57BL/6与ICR小鼠在博来霉素(BLM)致肺纤维化过程中的种属差异。方法 8周龄雌性C57BL/6小鼠19只,ICR小鼠16只,分别经尾静脉一次性注射BLM150mg/kg,观察每组小鼠体重、生存率及肺组织病理改变。结果①C57BL/6与ICR小鼠最低体重分别发生在静脉注射处置后的7d和5d,最低体重分别为注射前的65.46%和73.21%,两组间无显著的统计学差异。②C57BL/6与ICR小鼠的生存率分别为36.84%和56.25%,两组间存在显著的统计学差异。③C57BL/6小鼠BLM注射后28d,在胸膜下及血管周围形成广泛、稳定的间质纤维化病理改变,而ICR小鼠肺组织未见明显纤维化形成。C57BL/6小鼠肺纤维化病理评分明显高于ICR小鼠(P0.001)。结论 BLM诱导的肺纤维化作用在C57BL/6与ICR小鼠间存在着明显的种属差异。C57BL/6小鼠较ICR小鼠更适于复制博来霉素诱导的肺纤维化动物模型。     

Age of ovary determines remaining life expectancy in old ovariectomized mice

We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.     

Isolated deficiency of immunocytochemically detected somatostatin in Snell dwarf, but not in “little,” mice

Immunocytochemical staining of the neuropeptide somatostatin was evaluated in the brains of two growth hormone-deficient mouse mutants, Snell dwarf (dw/dw), and “little” (lit/lit). In Snell dwarf mice, in which GH is undetectable, an isolated somatostatin deficiency was observed in hypothalamic median eminence and in anterior periventricular hypothalamic neurons which are afferent to median eminence, compared to somatostatin staining in normal mice of the same strain (DW/?). Somatostatin staining in all other CNS areas in dwarfs was identical to that in normal mice. In contrast, in little mice, which exhibit 5–10% of normal GH levels, somatostatin expression was comparable between mutants and normal mice (LIT/?) in all CNS areas, including median eminence-afferent neurons in anterior periventricular hypothalamus. The results suggest that expression of somatostatin in hypophysiotropic CNS is dependent upon minimal pituitary GH secretion.     

Mouse models of uveal melanoma: Strengths,weaknesses, and future directions

Uveal melanoma is the most common primary malignancy of the eye, and a number of discoveries in the last decade have led to a more thorough molecular characterization of this cancer. However, the prognosis remains dismal for patients with metastases, and there is an urgent need to identify treatments that are effective for this stage of disease. Animal models are important tools for preclinical studies of uveal melanoma. A variety of models exist, and they have specific advantages, disadvantages, and applications. In this review article, these differences are explored in detail, and ideas for new models that might overcome current challenges are proposed.