Chemical mutagenesis of the mouse genome: an overview

The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a gene driven approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a phenotype driven approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.     

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

基因打靶定点突变秦川牛MSTN基因

Myostatin(MSTN,肌肉生长抑制素)基因属于TGF-β超家族,对骨骼肌的生长发育具有负调控作用。该基因的功能缺失,能够引起肉用动物的"双肌"表型,从而提高产肉率。基因打靶技术是制作转基因动物的常用方法。构建了两个置换型打靶载体pA2T-Mstn4.0和pA2T-Mstn3.2,通过同源重组将G938A突变点引入秦川牛MSTN基因第三外显子。电穿孔方法转染秦川牛胎儿成纤维细胞,经过600μg/mL G418和50nmol/L GCV的药物正负筛选,共得到170个药物抗性细胞克隆。对细胞克隆进行PCR、测序及Southern blotting鉴定,结果显示,第58号细胞克隆为发生了正确同源重组的中靶细胞。牛胎儿成纤维细胞中的MSTN基因的一条等位基因被成功改造。     

An essential gene mutagenesis screen across the highly conserved piebald deletion region of mouse chromosome 14

The piebald deletion complex is a set of overlapping chromosomal deficiencies on distal mouse chromosome 14. We surveyed the functional genetic content of the piebald deletion region in an essential gene mutagenesis screen of 952 genomes to recover seven lethal mutants. The ENU‐induced mutations were mapped to define genetic intervals using the piebald deletion panel. Lethal mutations included loci required for establishment of the left‐right embryonic axis and a loss‐of‐function allele of Phr1 resulting in respiratory distress at birth. A functional map of the piebald region integrates experimental genetic data from the deletion panel, mutagenesis screen, and the targeted disruption of specific genes. A comparison of several genomic intervals targeted in regional mutagenesis screens suggests that the piebald region is characterized by a low gene density and high essential gene density with a distinct genomic content and organization that supports complex regulatory interactions and promotes evolutionary stability. genesis 47:392–403, 2009. © 2009 Wiley‐Liss, Inc.     

ENU mutagenesis in the mouse: application to human genetic disease.

Genetic approaches in model organisms provide a powerful means by which to examine the biological basis of human diseases as well as the physiological processes that are affected by them. Although not without its drawbacks, the mouse has become the mammalian species of choice in studying the molecular basis of disease. Targeted mutagenesis approaches in the mouse have led to dramatic increases in our understanding of human disease processes. As a complement to these gene-driven studies, three developments have led to the reassessment of a phenotype-driven approach in the mouse--the accumulation of information that has emerged from human and mouse genome sequencing projects, the use of high-efficiency point mutagens such as N-ethyl-N-nitrosourea (ENU) and the application of systematic hierarchical screening protocols for the mouse. In this paper, progress with existing phenotypic screening programmes is discussed and opportunities for the development of new mouse disease models are presented.     

牛肌肉生长抑制素基因突变的遗传效应与育种应用

肌肉生长抑制素（myostatin,MSTN）基因主要在骨骼肌中表达,参与调控骨骼肌的生长发育。MSTN基因在不同物种中具有极强的进化保守性,同时还具有较多的突变多态性。在牛的不同品种中,存在不同位点的有义突变,突变型牛均表现为骨骼肌发达,呈现双肌表型,生长速度与产肉率显著提高。同时,该基因突变也引起显著的生理性遗传效应。对国内外肉牛的MSTN基因突变类型、突变后遗传效应及在肉牛育种应用等方面作了重点阐述,以期为我国地方品种肉牛改良和选育研究提供参考。     

肌肉生长抑制素调控动物骨骼肌生长机制的研究进展

肌肉生成抑制素（myostatin, MSTN）在动物机体骨骼肌的增殖、分化和生长中起着重要的负调控作用。MSTN基因的过表达会阻碍骨骼肌增殖分化及生长发育,而缺失或表达降低则会导致肌肉肥大,形成双肌现象（double muscle phenomenon, DMP）。MSTN能作用于多个基因及结合多种细胞因子广泛参与生理生化、物质代谢、病理调控等过程,在动物机体生长发育过程中扮演着重要的角色。本文将从MSTN基因的历史渊源、基因定位、时空表达特性、部分相关作用机制等方面进行论述,旨在对MSTN调控动物骨骼肌生长部分机制作梳理,以期为后期研究提供理论依据。     

A simple method to approximate gene content in large pedigree populations: application to the myostatin gene in dual-purpose Belgian Blue cattle

Gene content is the number of copies of a particular allele in a genotype of an animal. Gene content can be used to study additive gene action of candidate gene. Usually genotype data are available only for a part of population and for the rest gene contents have to be calculated based on typed relatives. Methods to calculate expected gene content for animals on large complex pedigrees are relatively complex. In this paper we proposed a practical method to calculate gene content using a linear regression. The method does not estimate genotype probabilities but these can be approximated from gene content assuming Hardy-Weinberg proportions. The approach was compared with other methods on multiple simulated data sets for real bovine pedigrees of 1 082 and 907 903 animals. Different allelic frequencies (0.4 and 0.2) and proportions of the missing genotypes (90, 70, and 50%) were considered in simulation. The simulation showed that the proposed method has similar capability to predict gene content as the iterative peeling method, however it requires less time and can be more practical for large pedigrees. The method was also applied to real data on the bovine myostatin locus on a large dual-purpose Belgian Blue pedigree of 235 133 animals. It was demonstrated that the proposed method can be easily adapted for particular pedigrees.     

A gene trap mutagenesis screen for genes underlying cellular response to the mood stabilizer lithium

Identifying the biological pathways mediating the action of a therapeutic compound may help the development of more specific treatments while also increasing our understanding of the underlying disease pathology. Salts of the metal lithium are commonly used as a front‐line mood stabilizing treatment for bipolar disorder. Lithium's action has been variously linked to inositol phosphate metabolism and the WNT/Glycogen Synthase Kinase 3β (GSK3β)/β‐Catenin signalling cascade, but, to date, little is known about which of these provides the principal therapeutic benefit for patients and, more specifically, which constituent genes, through presumed sequence variation, determine differences in patient response to treatment. Here, we describe a functional screen in which SH‐SY5Y neuroblastoma cells were randomly mutated through genomic integration of the pMS1 poly A ‘gene trap’ plasmid vector. Lithium normally induces differentiation of neuroblastoma cells, but a small proportion of mutated cells continued to proliferate and formed colonies. Rapid amplification of cDNA ends (RACE)‐PCR was used to identify the ‘trapped’ gene in each of these lithium‐resistant colonies. Heterozygous, gene trap integrations were identified within ten genes, eight of which are likely to produce loss‐of‐function mutations including MED10, MSI2 and three long intergenic non‐coding (LINC) RNAs. Both MED10 and MSI2 have been previously linked with WNT/GSK3β/β‐Catenin pathway function suggesting that this is an important mediator of lithium action in this screen. The methodology applied here provides a rapid, objective and economic approach to define the genetic contribution to drug action, but could also be readily adapted to any desired in vitro functional selection/screening paradigm.     

Lean and obese pig breeds exhibit differences in prenatal gene expression profiles of muscle development

Muscle development in domesticated animals is important for meat production. Furthermore, intramuscular fat content is an important trait of meat intended for consumption. Here, we examined differences in the expression of factors related to myogenesis, adipogenesis and skeletal muscle growth during fetal muscle development of lean (Yorkshire) and obese (Chenghua) pig breeds. At prenatal days 50 (d50) and 90 (d90), muscles and sera were collected from pig fetuses. Histology revealed larger diameters and numbers of myofibers in Chenghua pig fetuses than those in Yorkshire pig fetuses at d50 and d90. Yorkshire fetuses had higher serum concentrations of myostatin (d90), a negative regulator for muscle development, and higher mRNA expression of the growth hormone receptor Ghr (d90), myogenic MyoG (d90) and adipogenic LPL (d50). By contrast, Chenghua fetuses exhibited higher serum concentration of growth hormone (d90), and higher mRNA expression of myogenic MyoD (d90) as well as adipogenic PPARG and FABP4 (d50). Our results revealed distinct expression patterns in the two pig breeds at each developmental stage before birth. Compared with Chenghua pigs, development and maturation of fetal skeletal muscles may occur earlier in Yorkshire pigs, but the negative regulatory effects of myostatin may suppress muscle development at the later stage.     

Age-associated mitochondrial DNA deletions in mouse skeletal muscle: Comparison of different regions of the mitochondrial genome

The abundance of mitochondrial DNA (mtDNA) deletions has been shown to increase with age in a number of species and may contribute to the aging process. Estimating the total mtDNA deletion load of an individual is essential in evaluating the potential physiological impact. In this study, we compared three 5-kb regions of the mitochondrial genome: one in the major arc, one in the minor arc, and a third containing the light strand origin of replication. Through PCR analysis of mouse skeletal muscle, we have determined that not all regions produce equal numbers of age-associated deletions. There are, on average, twofold more detectable deletions in the major arc region than in the minor arc region. Deletions that result in the loss of the light strand origin of replication are rarely detected. Furthermore, the mechanism of deletion formation seems to be similar in both the major and minor arcs, with direct repeats playing an important, although not essential, role. © 1996 Wiley-Liss, Inc.     

Further characterization of a somatic cell hybrid panel: ten new assignments to the bovine genome

Thirty-six partially characterized hamster-bovine hybrid cell lines were used for the determination of synteny groups. Sixteen additional reference loci, selected for their coverage of the bovine genome, were analysed on these hybrid cells. This increases to 25 the number of synteny groups detected. This panel was then used to make synteny assignments for 10 additional loci, eight by Southern blotting (COL1A1, COL1A2, FAS, CTSB, CTSL, CHRNG, HEXB and HTR1A) and two by polymerase chain reaction (PCR) amplification (HRH1 and ETH1112), These loci were assigned to international synteny groups U12 (HRH1), U13 (COL1A2), U17 (CHRNG), U21 (COL1A1, FAS), U29 (ETHI1112), to chromosome 20 (U14 or U25) for HEXB and HTR1A, and to the same local synteny group (A), which is probably U18, for CTSB and CTSL. For three loci already mapped in humans (COL1A1, COL1A2 and CHRNG), the present results are in accordance with the predictions based on comparative mapping between the human and bovine species.     

A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics.

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.     

Profiling of the yak skeletal muscle tissue gene expression and comparison with the domestic cattle by genome array

Of all the mammals of the world, the yak lives at the highest altitude area of more than 3000 m. Comparison between yak and cattle of the low-altitude areas will be informative in studying animal adaptation to higher altitudes. To investigate the molecular mechanism involved in meat quality differences between the two Chinese special varieties Qinghai yak and Qinchuan cattle, 12 chemical–physical characteristics of the longissimus dorsi muscle related to meat quality were compared at the age of 36 months, and the gene expression profiles were constructed by utilizing the bovine genome array. Significant analysis of microarrays was used to identify the differentially expressed genes. Gene ontology and pathway analysis were performed by a free Web-based Molecular Annotation System 2.0. The results reveal ~11 000 probes representing about 10 000 genes that were detected in both the Qinghai yak and Qinchuan cattle. A total of 1922 genes were shown to be differentially expressed, 633 probes were upregulated and 1259 probes were downregulated in the muscle tissue of Qinghai yak that were mainly involved in ubiquitin-mediated proteolysis, muscle growth regulation, glucose metabolism, immune response and so on. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. Further analysis implied that animals living at a high altitude may supply energy by more active protein catabolism and glycolysis compared with those living in the plain areas. Our results establish the groundwork for further studies on yaks’ meat quality and will be beneficial in improving the yaks’ breeding by molecular biotechnology.     

Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene

Myostatin (GDF-8, MSTN) is a member of trans- forming growth factors (TGF-β) superfamily, which was first described by McPherron et al. in 1997[1]. Myostatin appears to act as a negative regulator of muscle development and controls not only fibre size but also fibre number[2,3]. Mutations in the third exon of the myostatin gene have been shown to cause dou- ble muscling in cattle[4]. By knocking out the gene of myostatin in mice, they were able to show that the transgenic mice developed …     

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.     

Haploinsufficiency of myostatin protects against aging‐related declines in muscle function and enhances the longevity of mice

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28–30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN^+/− and MSTN^−/− mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN^+/+ and MSTN^−/− mice, MSTN^+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.