Fancm基因敲除小鼠构建及其表型分析

目的构建范可尼贫血通路Fancm基因敲除小鼠,研究Fancm基因缺失对小鼠生理功能,特别是雄性生殖器官的影响。方法采用CRISPR/Cas9技术,获得Fancm基因敲除小鼠。分析FANCM蛋白在野生型和Fancm^-/-小鼠睾丸组织中的表达。统计Fancm^-/-小鼠的出生率、体重、性别比例及子代生育情况,分析血液常规指标。组织形态学研究雄性Fancm^-/-小鼠睾丸生理病理表型。结果敲除Fancm基因ATG区域,获得稳定遗传的C57BL/6背景Fancm^-/-小鼠。Fancm^-/-小鼠睾丸中FANCM蛋白表达完全丢失。Fancm^-/-小鼠无明显的胚胎致死现象,但雌性Fancm-/-小鼠数目显著少于雄性Fancm^-/-小鼠。同窝Fancm^-/-小鼠比较野生型体重无明显区别,部分血常规指标有显著性差异。Fancm^-/-小鼠有明显的生殖能力缺陷。雄性Fancm^-/-小鼠睾丸有显著的发育缺陷,其生精细胞凋亡增加、细胞周期阻滞,影响睾丸发育与精子的生成。结论成功获得稳定遗传C57BL/6背景Fancm^-/-小鼠,Fancm基因参与小鼠的生长发育,特别是雄性生殖器官功能的维持及调控。     

Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice

The cleavage-product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer's disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down-regulated mouse APP protein levels (APP-siRNA) were identified using an APP plasmid-siRNA co-transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP-siRNAs into the ventricular system for 2 weeks also down-regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2-CA3 regions of the hippocampus. Mice treated with the most active APP-siRNA showed a significant reduction in spontaneous alternation rate in the Y-maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus-dependent spatial working memory.     

Prediction of siRNA knockdown efficiency using artificial neural network models

Selective knockdown of gene expression by short interference RNAs (siRNAs) has allowed rapid validation of gene functions and made possible a high throughput, genome scale approach to interrogate gene function. However, randomly designed siRNAs display different knockdown efficiencies of target genes. Hence, various prediction algorithms based on siRNA functionality have recently been constructed to increase the likelihood of selecting effective siRNAs, thereby reducing the experimental cost. Toward this end, we have trained three Back-propagation and Bayesian neural network models, previously not used in this context, to predict the knockdown efficiencies of 180 experimentally verified siRNAs on their corresponding target genes. Using our input coding based primarily on RNA structure thermodynamic parameters and cross-validation method, we showed that our neural network models outperformed most other methods and are comparable to the best predicting algorithm thus far published. Furthermore, our neural network models correctly classified 74% of all siRNAs into different efficiency categories; with a correlation coefficient of 0.43 and receiver operating characteristic curve score of 0.78, thus highlighting the potential utility of this method to complement other existing siRNA classification and prediction schemes.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

The assessment of arboreal insect fauna: comparisons of knockdown sampling and faunal lists

Abstract. 1. A comparison is made between the fauna of six British tree species sampled by pyrethrum knockdown and the faunal lists in the literature for the same tree species.
2. Conspecific trees vary both in abundance of arthropods and their proportional distribution across taxa, but there are important overriding differences between tree species.
3. The percentage similarity in faunal composition between tree species reflects the ecological specificity of the arthropod groups used for the comparison.
4. The similarity in species composition between tree species is least for phytophages, the guild most closely associated with particular tree species, The two species of Salix are most similar in their fauna for most guilds.
5. About 40% of the entirely phytophagous species in the faunal lists for native trees were found in the knockdown samples.
6. The proportion of individuals of predominantly phytophagous taxa collected that belong to the relevant faunal lists ranges from 0.39 to 0.99.
7. Those species found in the knockdown samples which are not included in faunal lists none the less contribute to the trophic web of the tree.
8. The relative species richness of arboreal faunas assessed from knockdown samples parallels that derived from faunal lists.
9. The two approaches to the categorization of arboreal faunas, knockdown sampling and faunal lists, provide comparable data.     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.     

Reproducible and inducible knockdown of gene expression in mice

RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies.     

Phenotypes of SERCA and PMCA knockout mice

P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction.     

The broken mouse: the role of development, plasticity and environment in the interpretation of phenotypic changes in knockout mice

With the advent of gene knockout technology has arisen the problem of how to interpret the resulting phenotypic changes in mice lacking specific genes. This problem is especially relevant when applied to behavioral phenotypes of knockout mice, which are difficult to interpret. Of particular interest are the roles of development and compensatory changes, as well as other factors, such as the influence of the gene knockout on nearby genes, the effect of the genetic background strain, maternal behavioral influences, and pleiotrophy.     

基因敲除小鼠技术的研究进展

基因敲除小鼠技术的建立和发展使得人们为研究基因的功能和寻找新的治疗人类疾病的靶点提供了强有力的支持。基因打靶和基因捕获是两种通过胚胎干细胞(Embryonicstemcell,ESC)构建基因敲除小鼠的技术。基因打靶通过同源重组替换内源基因从而敲除目的基因,而基因捕获则有启动子捕获和polyA捕获两种方法对目的基因进行敲除。近年来,有许多新的基因敲除技术不断被开发出来,包括Cre/loxP系统、CRISP/Cas9系统以及最新的ZFN技术和TAILEN技术,都有望取代传统基因敲除手段。文中简要阐述了如今新出现的几种基因敲除小鼠技术。     

Wound-healing studies in transgenic and knockout mice

Injury to the skin initiates a cascade of events including inflammation, new tissue formation, and tissue remodeling, that finally lead to at least partial reconstruction of the original tissue. Historically, animal models of repair have taught us much about how this repair process is orchestrated and, over recent years, the use of genetically modified mice has helped define the roles of many key molecules. Aside from conventional knockout technology, many ingenious approaches have been adopted, allowing researchers to circumvent such problems as embryonic lethality, or to affect gene function in a tissue-or temporal-specific manner. Together, these studies provide us with a growing source of information describing, to date, the in vivo function of nearly 100 proteins in the context of wound repair. This article focuses on the studies in which genetically modified mouse models have helped elucidate the roles that many soluble mediators play during wound repair, encompassing the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) families and also data on cytokines and chemokines. Finally, we include a table summarizing all of the currently published data in this rapidly growing field. For a regularly updated web archive of studies, we have constructed a Compendium of Published Wound Healing Studies on Genetically Modified Mice which is available at http://icbxs.ethz.ch/members/grose/woundtransgenic/home.html.     

Dynamic instability of the major urinary protein gene family revealed by genomic and phenotypic comparisons between C57 and 129 strain mice

Background  

The major urinary proteins (MUPs) of Mus musculus domesticus are deposited in urine in large quantities, where they bind and release pheromones and also provide an individual 'recognition signal' via their phenotypic polymorphism. Whilst important information about MUP functionality has been gained in recent years, the gene cluster is poorly studied in terms of structure, genic polymorphism and evolution.     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.     

Generation and characterization of Rac3 knockout mice

Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.