A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.     

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.     

C1q deficiency and autoimmunity: the effects of genetic background on disease expression

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 x C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp(+/+) strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp(+/+) animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp(+/+) mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp(+/+) strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.     

Mouse phenome research: implications of genetic background

Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The "genetic background" (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticus subspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.     

The Arabidopsis SeedGenes Project

The SeedGenes database (http://www.seedgenes.org) presents molecular and phenotypic information on essential, non-redundant genes of Arabidopsis that give a seed phenotype when disrupted by mutation. Experimental details are synthesized for efficient use by the community and organized into two major sections in the database, one dealing with genes and the other with mutant alleles. The database can be queried for detailed information on a single gene to create a SeedGenes Profile. Queries can also generate lists of genes or mutants that fit specified criteria. The long-term goal is to establish a complete collection of Arabidopsis genes that give a knockout phenotype. This information is needed to focus attention on genes with important cellular functions in a model plant and to assess from a genetic perspective the extent of functional redundancy in the Arabidopsis genome.     

<Emphasis Type="Italic">Cis</Emphasis>-acting variation in the expression of a high proportion of genes in human brain

Much of the genetic component of human phenotypic diversity, including susceptibility to disease, is proposed to be the result of cis-acting influences on gene expression. If this hypothesis is correct, it implies that cis-acting regulatory variation is a common phenomenon. However, direct evidence in support of this view is scarce. We have applied highly quantitative measures of allele-specific expression in order to screen an average of 19 informative subjects (range 8-26) for the presence of common cis-acting influences on the expression of 15 genes by using RNA derived from human brain. We found that, in seven of the 15 assayed genes, at least one individual exhibited relative differences in allelic expression of 20% or more and, in one gene (DTNBP1), allelic expression differences exceeded 50%. These results suggest that cis-acting variation in gene expression commonly occurs in native tissue and hence provide empirical support for the hypothesis that this is potentially an important mechanism underlying human phenotypic diversity.     

The pleiotropic phenotype of Apc mutations in the mouse: allele specificity and effects of the genetic background

Familial adenomatous polyposis (FAP) is a human cancer syndrome characterized by the development of hundreds to thousands of colonic polyps and extracolonic lesions including desmoid fibromas, osteomas, epidermoid cysts, and congenital hypertrophy of the pigmented retinal epithelium. Afflicted individuals are heterozygous for mutations in the APC gene. Detailed investigations of mice heterozygous for mutations in the ortholog Apc have shown that other genetic factors strongly influence the phenotype. Here we report qualitative and quantitative modifications of the phenotype of Apc mutants as a function of three genetic variables: Apc allele, p53 allele, and genetic background. We have found major differences between the Apc alleles Min and 1638N in multiplicity and regionality of intestinal tumors, as well as in incidence of extracolonic lesions. By contrast, Min mice homozygous for either of two different knockout alleles of p53 show similar phenotypic effects. These studies illustrate the classic principle that functional genetics is enriched by assessing penetrance and expressivity with allelic series. The mouse permits study of an allelic gene series on multiple genetic backgrounds, thereby leading to a better understanding of gene action in a range of biological processes.     

Beyond mean allelic effects: A locus at the major color gene MC1R associates also with differing levels of phenotypic and genetic (co)variance for coloration in barn owls

The mean phenotypic effects of a discovered variant help to predict major aspects of the evolution and inheritance of a phenotype. However, differences in the phenotypic variance associated to distinct genotypes are often overlooked despite being suggestive of processes that largely influence phenotypic evolution, such as interactions between the genotypes with the environment or the genetic background. We present empirical evidence for a mutation at the melanocortin‐1‐receptor gene, a major vertebrate coloration gene, affecting phenotypic variance in the barn owl, Tyto alba. The white MC1R allele, which associates with whiter plumage coloration, also associates with a pronounced phenotypic and additive genetic variance for distinct color traits. Contrarily, the rufous allele, associated with a rufous coloration, relates to a lower phenotypic and additive genetic variance, suggesting that this allele may be epistatic over other color loci. Variance differences between genotypes entailed differences in the strength of phenotypic and genetic associations between color traits, suggesting that differences in variance also alter the level of integration between traits. This study highlights that addressing variance differences of genotypes in wild populations provides interesting new insights into the evolutionary mechanisms and the genetic architecture underlying the phenotype.     

Genetic variations strongly influence phenotypic outcome in the mouse retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2(-nb1) at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases.     

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner

Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood.     

Genetic background determines metabolic phenotypes in the mouse

To evaluate the contribution of genetic background to phenotypic variation, we compared a large range of biochemical and metabolic parameters at different ages of four inbred mice strains, C57BL/6J, 129SvPas, C3HeB/FeJ, and Balb/cByJ. Our results demonstrate that important metabolic, hematologic, and biochemical differences exist between these different inbred strains. Most of these differences are gender independent and are maintained or accentuated throughout life. It is therefore imperative that the genetic background is carefully defined in phenotypic studies. Our results also argue that certain backgrounds are more suited to study a given physiologic phenomenon, as distinct mouse strains have a different propensity to develop particular biochemical, hematologic, and metabolic abnormalities. These genetic differences can furthermore be exploited to identify new genes/proteins that contribute to phenotypic abnormalities. The choice of the genetic background in which to generate and analyze genetically engineered mutant mice is important as it is, together with environmental factors, one of the most important contributors to the variability of phenotypic results.     

基因剔除小鼠研究的新进展

基因剔除小鼠是活体内研究基因及蛋白质功能的理想动物模型。传统的基因剔除技术存在表型分析复杂、周期长、操作繁杂等缺点,如何更科学、更方便地建立基因剔除小鼠模型成为目前使该技术更好地应用于医学研究的关键。本文从胚胎干细胞（ES细胞）的遗传背景、组织特异性基因表达基因剔除小鼠及基因剔除技术上的革新几方面介绍了目前基因剔除小鼠研究的一些最新进展。     

A gene's ability to buffer variation is predicted by its fitness contribution and genetic interactions

Background

Many single-gene knockouts result in increased phenotypic (e.g., morphological) variability among the mutant''s offspring. This has been interpreted as an intrinsic ability of genes to buffer genetic and environmental variation. A phenotypic capacitor is a gene that appears to mask phenotypic variation: when knocked out, the offspring shows more variability than the wild type. Theory predicts that this phenotypic potential should be correlated with a gene''s knockout fitness and its number of negative genetic interactions. Based on experimentally measured phenotypic capacity, it was suggested that knockout fitness was unimportant, but that phenotypic capacitors tend to be hubs in genetic and physical interaction networks.

Methodology/Principal Findings

We re-analyse the available experimental data in a combined model, which includes knockout fitness and network parameters as well as expression level and protein length as predictors of phenotypic potential. Contrary to previous conclusions, we find that the strongest predictor is in fact haploid knockout fitness (responsible for 9% of the variation in phenotypic potential), with an additional contribution from the genetic interaction network (5%); once these two factors are taken into account, protein-protein interactions do not make any additional contribution to the variation in phenotypic potential.

Conclusions/Significance

We conclude that phenotypic potential is not a mysterious “emergent” property of cellular networks. Instead, it is very simply determined by the overall fitness reduction of the organism (which in its compromised state can no longer compensate for multiple factors that contribute to phenotypic variation), and by the number (and presumably nature) of genetic interactions of the knocked-out gene. In this light, Hsp90, the prototypical phenotypic capacitor, may not be representative: typical phenotypic capacitors are not direct “buffers” of variation, but are simply genes encoding central cellular functions.     

Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.     

Environmental dependency of gene knockouts on phenotype microarray analysis in Escherichia coli

Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray? (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.     

Genetics and cardiovascular system: influence of human genetic variants on vascular function

Candidate gene association studies in cardiovascular diseases have provided evidence on the molecular basis of phenotypic differences between individuals. The comprehension of how inherited genetic variants are able to affect protein functions has increased the knowledge of how genes interact with environment in order to modulate a particular phenotype. Although it is known that the human genome contains more than 10 million SNPs, only a minor part of them are supposed to be functional. A causative SNP in a particular gene may confer a small to moderate effect in complex phenotypes, such as functions important to cardiovascular homeostasis. This paper is a selective review of the literature on the evidence for interactions between vascular function and naturally occurring genetic variants in endothelial nitric oxide synthase (eNOS) and beta-2 adrenergic receptor (ADRB2), two genes among those influencing vascular phenotype and examples for which there is a strong evidence base. eNOS and ADRB2 will be characterized, as well as the mechanisms by which the enzyme and the receptor work to control vascular responses will be described. Understanding the molecular mechanisms underlying gene-mediated vascular function and their modification by genetic variants is expected to result in a better comprehension about individual's phenotypic differences.     

Cloning of Flagellar Genes in Chlamydomonas Reinhardtii by DNA Insertional Mutagenesis

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.     

Interpretation of phenotype in genetically engineered mice.

BACKGROUND AND PURPOSE: In mice, genetic engineering involves two general approaches-addition of an exogenous gene, resulting in transgenic mice, and use of knockout mice, which have a targeted mutation of an endogenous gene. The advantages of these approaches is that questions can be asked about the function of a particular gene in a living mammalian organism, taking into account interactions among cells, tissues, and organs under normal, disease, injury, and stress situations. METHODS: Review of the literature concentrating principally on knockout mice and questions of unexpected phenotypes, lack of phenotype, redundancy, and effect of genetic background on phenotype will be discussed. CONCLUSION: There is little gene redundancy in mammals; knockout phenotypes exist even if none are immediately apparent; and investigating phenotypes in colonies of mixed genetic background may reveal not only more phenotypes, but also may lead to better understanding of the molecular or cellular mechanism underlying the phenotype and to discovery of modifier gene(s).