deletions , point mutations , amplifications , insertions , inversions and translocations can alter the dna sequence . transmission from parent to offspring results in fixation of these mutations within a population . as schematized in the poster , mice caught in the wild can contain one of four bases ( a , c , g or t ) at a given locus ; a female and male are initially mated in the laboratory to produce offspring . inbreeding by brother - sister matings for 20 consecutive generations creates inbred strains ( see box 1 for definition of terms ) of mice , and each individual strain is virtually homozygous for every gene in the genome ( isogenic ) ( silver , 1995 ) . inbred strains have thus captured a portion of the natural variation present in wild mice . the mouse genome database describes the origins and characteristics of inbred strains and is a comprehensive database for information about the mouse ( www.informatics.jax.org ) . given the large number of inbred strains , it is estimated that these collective resources now exhibit more variation than that present in the human population . natural variation accounts for the differences in phenotype exhibited among inbred strains ( as well as between individuals ) . sequence differences can alter transcripts and proteins by altering their functional properties , as well as the timing , level and site(s ) of expression in the body . monogenic traits are controlled by only one gene , whereas complex traits are controlled by two or more genes . intercrosses and backcrosses between inbred strains exhibiting the extremes of a specific phenotype permit a general localization of genes influencing the trait by matching phenotype to genotype . modifier genes , namely genes that can modify the phenotype of an already existing mutation or disease allele ; thus , a disease can manifest as mild or severe , depending on the influence of modifier alleles ( nadeau , 2003 ) . the chromosomal location of each type of gene can be refined through the use of consomic , conplastic , congenic , recombinant inbred ( ri ) and/or recombinant congenic ( rc ) strains ( roberts et al . , 2007 ) . an array of possible forms of a gene , which can each cause different phenotypic effects . the mating of a heterozygous individual with one of its inbred parents , or with an individual of the same genotype as one of its inbred parents , to follow the inheritance of alleles and phenotypes . a strain derived by backcrossing an allele or mutation of interest onto the background of a different strain for at least ten generations to achieve allelic transfer . a strain that carries the mitochondrial genome from a donor strain and the nuclear genome of a recipient strain . conplastic strains are made by intercrossing two inbred strains , followed by sequential crosses of females to recipient males . a strain that carries one pair of homologous whole chromosomes from a donor strain on the genetic background of a recipient strain . a complete set of consomic strains is 21 , which includes those with each of the 19 autosome pairs , the x chromosome pair and the y chromosome . a genetic analysis that proceeds from phenotype to genotype by positional cloning or candidate gene analysis . a cross between two individuals with the same heterozygous genotype ( usually a brother by sister mating ) , to follow the inheritance of alleles and phenotypes . a set of strains derived from intercrossing two inbred strains , followed by a small number of backcross generations prior to inbreeding . a subset of the donor genome remains present on the background of the recipient strain . a set of strains derived from intercrossing two inbred strains , then brother by sister mating their f2 offspring for at least 20 generations to derive new inbred strains . each strain represents a random mixture of genes from the two parental strains that are fixed in the new inbred strain . a genetic analysis that proceeds from genotype to phenotype by genetic engineering techniques , such as homologous recombination in embryonic stem ( es ) cells . an electronic version of this poster is available online . natural variation can be used to identify genes involved in a variety of quantitative traits and diseases ( hunter and crawford , 2008 ) . already established and developing resources are designed to mimic the diversity present in human populations . the collaborative cross ( cc ) is a combination of the genotypes of eight inbred strains , specifically chosen because of the diversity of their genomes ( only four strains are shown in the poster for simplicity ) ( churchill et al . , 2004 ) . each of the eight inbred strains that contribute to the cc is being sequenced , so that the molecular genetic contribution of each chromosomal region will be known by determining the genotype of each cc strain using the mouse diversity chip ( yang et al . , 2009 ) . because the molecular composition of each cc strain will be known , phenotyping each strain for a trait such as immune response , blood glucose level , bone density or social behavior will allow researchers to determine a refined chromosomal location for quantitative trait loci ( qtls ) affecting the trait of interest using in silico methods . an advantage of the cc is that all data are cumulative , allowing for cross - referencing of phenotype and genotype databases derived from the cc strains . heterogeneous stock ( hs ) populations also represent contributions from eight inbred strains ; however , hs populations are maintained by random matings of mice within a colony ( valdar et al . , 2006 ) . therefore , although the molecular contribution from the eight progenitor inbred strains is known , new inbred strains are not created . thus , each individual hs mouse has a unique combination of alleles ( see poster ) . hs mice are designed to contain random variation within a population that would be similar to that found in the human population , allowing for a controlled molecular knowledge of the alleles that contribute to each mouse . because each individual derived from the hs cross is unique , the molecular component of each mouse must be genotyped , and each mouse must be phenotyped to identify genes affecting the trait of interest . the benefit of these novel resources is the ability to map a trait of interest to a small chromosomal region , and ultimately to identify the genes and pathways responsible so as to better prevent and treat human disease . current sequencing efforts for the human genome are revealing a large number of genetic alterations that occur from generation to generation . the nature of these  private mutations and their impact on phenotype has yet to be fully evaluated . such individual variation contributes to a phenotype by uniquely affecting the individual who carries such changes . these types of changes can be evaluated in the mouse by using reverse genetics or transgenesis . the ability to introduce a mutation by design is termed reverse genetics . of all experimental organisms , fluent reverse genetics is currently only available in escherichia coli , yeast and the mouse , owing to the remarkable renewing properties of mouse embryonic stem cells ( mescs ) ( soriano , 1995 ) . this feature is one of the main reasons for the pre - eminence of the mouse as the leading mammalian model system ( oliver et al . , 2007 ) . mescs are derived from early - stage embryos and can be grown in culture yet retain the ability to contribute to the formation of a mouse on reintroduction into an early - stage host embryo  usually at the blastocyst stage , as illustrated in the poster . because mescs can be grown in culture , the mouse genome can be engineered by transfection with dna constructs that have been engineered in vitro to carry a specifically designed mutation ( the insertion of loxp sites is shown in the poster ) . the dna construct can be precisely integrated into the mouse genome via homologous recombination , so that the existing dna sequence is altered exactly by design ( capecchi , 1989 ) . alternatively , the gene can be disrupted by random or transpositional  gene - trapping mutagenesis ( evans et al . , 1997 ) . gene trapping varies from targeted mutagenesis in that a construct carrying a reporter gene can insert into a gene , disrupting its function . the sequence flanking the insertion is determined subsequently , and the expression of the reporter can allow for the assessment of where the gene is expressed . a disadvantage is that the insertions might not disrupt the gene , or that they have the potential to affect flanking genes owing to their position . for either targeted mutations or gene - trapped mutations , transfected mesc clones can be selected after screening for disrupted alleles in the gene of interest . after injection into a blastocyst , the selected mesc clone mixes into the host embryo to contribute to a chimera composed of host and mutated cells ; this chimeric adult can propagate the mesc mutation by germline transmission to create a genetically engineered mouse strain . the ability to engineer the mouse genome via mescs stimulated the development of sophisticated genome engineering and gene expression strategies ( glaser et al . , 2005 ) . chief among these is conditional mutagenesis based on the use of cre - loxp site - specific recombination ( branda and dymecki , 2004 ) . two 34-bp loxp sites are introduced into a chosen gene , either by gene targeting or trapping , so that normal gene expression is undisturbed . floxed gene is mutagenized upon exposure to cre recombinase , which mediates dna recombination between the loxp sites . the mutagenic event can be controlled in space and time by regulating cre recombinase expression and/or activity ( schnutgen et al . , 2006 ) . for example , it could be that complete elimination of a gene of interest results in embryonic lethality due to a requirement in embryogenesis ; however , mating a conditional allele of this gene to a cre expresser during adulthood would result in the elimination of the gene long after it is required for embryonic development , allowing the researcher to investigate the function of the gene in the adult . a growing panel of mouse lines that express cre recombinase in specific cell types , termed the  cre zoo , is being developed to achieve spatial precision in mutagenesis ( see transgenics section ) . additionally , temporal regulation of cre recombinase activity can be achieved by expressing cre as a fusion protein with a steroid receptor ligand - binding domain , usually the mutant estrogen domain termed thus , crossing a cre driver mouse with a mouse carrying a floxed gene enables conditional mutagenesis in an inducible way . a gene that does not inherently belong to the organism , but is introduced into its genome from an outside source is called a transgene . transgenesis can be achieved in mice by the microinjection of manipulated dna into the pronucleus of a fertilized egg ( palmiter and brinster , 1985 ) . the dna inserts at random into the genome , and if it does so prior to cell division , it can contribute to all cells of the organism . exogenous transposons as well as engineered or targeted mutations are also transgenes ; however , these are not examples of microinjection transgenics . to obtain ova for microinjection , female mice must be superovulated using hormone injections to obtain a maximum number of embryos after mating to males . fertilized eggs are harvested and injected with linearized dna constructs in the laboratory , then surgically implanted into the oviducts of foster mothers that are the subsequent liveborn mice represent  founders carrying the transgene ; these mice must be analyzed for the presence or absence of the exogenous dna . for example , neomycin allows cells to grow in media that contains g418 , and hypoxanthine phosphoribosyltransferase ( hprt ) allows growth in medium containing hypoxanthine , aminopterin and thymidine ( hat ) . growing the cells in such media is required , but repeated selection can be detrimental to the cells ( plagge et al . , 2000 ) . furthermore , the presence of internal promoters required to drive the expression of selection cassettes can influence the expression of flanking genes . this can be achieved by the use of another site - specific recombination system derived from yeast : the flp recombinase catalyzes recombination at frt sites ( farley et al . , 2000 ) . the most commonly used mescs are ab2.2 , which are derived from the 129/ola inbred strain , and jm8 , which are derived from the c57bl/6n inbred strain . the availability of knockouts on either strain background reduces the likelihood that modifier genes will affect the phenotype after mice are made . however , each strain background has different germline transmission rates ( pettitt et al . , 2009 ) . germline transmission of the mesc genetic component is required to obtain mice that carry the construct . however , aneuploidy of the mescs can preclude the ability of the clone to achieve germline transmission . obtaining early passage mescs and reducing the manipulations required to generate the desired construct can make germline transmission more likely . human disease can also be caused by lesions other than deletions that alter gene function . for example , point mutations can be modeled using a  knock - in , whereby the precise lesion found in the human is introduced into the mouse using a two - step procedure in mescs ( plagge et al . , 2000 ) . alternatively , a transgene expressing the human disease mutation can be introduced into the knockout background . the first application of mouse transgenesis was a physiological one : the introduction of human growth hormone resulted in increased size ( palmiter et al . , 1982 ) . subsequent applications of transgenesis were to express a gene under the control of exogenous regulatory elements , or to drive a reporter gene under the control of regulatory elements . such experiments have been very powerful for cancer studies , as well as for understanding mechanisms of gene regulation . shown in the poster is an example of a cre driver mouse : a promoter element is used to drive expression of cre recombinase in a spatial- or temporal - specific pattern . as explained earlier , in the reverse genetics section , cre drivers are required for conditional deletion of a region of dna from mice that contain loxp sites . other applications of transgenics are to express a bacterial artificial chromosome ( bac ) or yeast artificial chromosome ( yac ) in the mouse , for gene rescue or for  humanization ( expression of a human gene or genomic region in the mouse ) . there are several problems with microinjection transgenics , which must be understood for their proper use . because integration is random , endogenous regulatory elements can influence the expression of the transgene.the linearized dna commonly integrates as a head - to - tail concatamer in copy numbers that vary from one to > 200 per haploid genome . copy number influences the level of expression , and copies can be lost in subsequent generations by gene conversion.if the dna integrates late in embryonic cell division , the founder mice can be mosaic , and must be bred to achieve contribution of the transgene to all cells . this is a common problem for large transgenes such as bacs.integration can result in an insertional mutation , as demonstrated in the transposon section ( see below ) . because integration is random , endogenous regulatory elements can influence the expression of the transgene . the linearized dna commonly integrates as a head - to - tail concatamer in copy numbers that vary from one to > 200 per haploid genome . copy number influences the level of expression , and copies can be lost in subsequent generations by gene conversion . if the dna integrates late in embryonic cell division , the founder mice can be mosaic , and must be bred to achieve contribution of the transgene to all cells . integration can result in an insertional mutation , as demonstrated in the transposon section ( see below ) . recent techniques using site - specific recombinases obtained from bacteriophages have been developed to alleviate these concerns . these include the use of  docking sites for site - specific integrases so that the effect of position and copy number is reduced , thus eliminating the requirement for multiple founders ( branda and dymecki , 2004 ) . an adaptation of cre - loxp conditional expression uses knock - in alleles to overcome many of the disadvantages of classical transgenics . a gene can be placed under the control of a ubiquitous promoter ( driving the expression of the gene in all cells at all times ) or self promoter ( driving the expression of the gene under the control of its normal promoter ) , but transcriptional stop sequences , which are flanked by loxp sites , can be inserted to block its expression . when the animal is mated to a cre expresser , or cre is spatially introduced ( as in viral cre delivery ) , recombination occurs between the loxp sites , allowing for deletion of the stop cassette , inducing expression of the gene in a tissue or site of interest ( zadelaar et al . , this application is powerful for cancer studies , because expression of a single copy gene is precisely regulated by time and location ; it can be designed to allow expression in a single cell ( marumoto et al . , 2009 ) . in forward genetics approaches , many agents , including chemicals , radiation and viruses , are used to disrupt genes to identify their functions and the diseases associated with them . the most powerful mutagen for forward genetic screens in mice is the alkylating agent n - ethyl - n - nitrosourea ( enu ) , which can produce mutation rates as high as 1.510 per mutagenized genome in male mouse spermatogonial stem cells ( russell et al . , 1979 ; guenet , 2005 ) . enu primarily produces point mutations , which include loss - of - function , gain - of - function , and super - active and partially active coding region mutations , as well as non - coding rna and regulatory mutations . after treatment with enu , male mice are mated in genetic screens designed to uncover mutations of interest ( justice , 2000 ) . dominant mutations are isolated by their phenotype in the first generation of breeding ( hrabe de angelis and balling , 1998 ) . mutations that result in visible phenotypes , such as changes in the coat , morphology or movement , are simple to detect . more advanced phenotype screening methods for behavior , hematology , pain perception and biochemistry have uncovered many previously unknown dominant mutations . screens for modifying mutations are likely to be the most common use of forward genetics in the future , because they are a powerful method for identifying disease suppressors ( carpinelli et al . , 2004 ) . in a modifier screen , a new unknown dominant mutation present in the enu - treated male gamete is isolated by its ability to modify ( either by enhancing or suppressing ) a known recessive or dominant phenotype that is produced by a mutation carried by a female mouse with which the male is mated , or that is present as a homozygous viable trait in the strain background . here , the idea of complex traits is taken to the extreme : instead of relying on natural variation , potent mutations are induced in dna , and the animal reveals important interactions . screens for recessive mutations using three - generation pedigree breeding schemes or using balancer chromosomes have been carried out ( kasarskis et al . , 1998 ; herron et al . , 2002 ; kile et al . , 2003 although recessive screens require more breeding time , their use has produced mutations to understand developmental pathways , immunology and responses to infection ( beutler et al . a mutation is mapped to a molecular interval , and then genes are sequenced to identify lesions caused by enu treatment and not repaired . banks of sperm and dna samples from mutagenized males are useful for identifying point mutations in desired genes ( coghill et al . , 2002 ) . the identification of point mutations is now straightforward owing to advances in sequencing technology and in mutation detection . once a mutation is identified , the affected gene must be confirmed by a second mutation , perhaps by a gene knockout , or by a rescue of the mutant phenotype using a transgene . transposable elements are discrete pieces of dna that can  jump around in the genome of a living organism . for each dna transposon , a corresponding protein , called a transposase , mediates the jumping . two exogenous transposon - transposase duos have proven to be powerful forward genetic mutagens in the mouse : sleeping beauty ( sb ) , which was a non - functioning system that has been resurrected from salmonid fish , and piggybac , which is native to the cabbage looper moth ( bestor , 2005 ; horie et al . , 2010 ) . unlike enu mutagenesis , which requires large amounts of sequencing to pinpoint each tiny change , transposable elements are powerful because their sequence is known , so when a transposon insertion mutates a gene , the transposon sequence provides a each of these elements works by a  cut - and - paste mechanism so that the transposase ( represented by scissors in the poster ) cuts the transposon out of one location , allowing the transposon to hop to a new location . the transposon is flanked by direct repeats ( represented by arrowheads ) , which are required for transposition . in its new location , the transposon can enhance gene expression ( green arrow ) , disrupt gene expression ( stop sign ) , or have no effect on gene expression . the most powerful application of the transposon systems in mice has been to identify genes that promote cancer ( collier and largaespada , 2007 ) . the system has applications outside mutagenesis , because the transposons can be engineered to deliver dna cargo to many locations in the genome using the direct repeat sequences . genome - wide association studies ( gwas ) are continually identifying a growing number of loci that are associated with human diseases [ see the catalog compiled by the national human genome research institute ( nhgri ) at www.genome.gov/gwastudies/index.cfm ] , increasing the need to investigate the function of these disease alleles in vivo . furthermore , whole - genome sequencing is just beginning to reveal what might be a multitude of rare , individual mutations that underlie human disease and other complex traits ( manolio et al . , 2009 ) . the only means to understand biological processes that occur in cells and physiological processes that occur in whole organisms is to recapitulate alleles associated with disease in a controlled setting , and to explore the effects of selected mutations and/or variants , both individually and in combination . the mouse is the only mammalian system that currently has the resources as well as the technology to fulfill this challenge . the combination of forward and reverse genetics , along with transgenesis and the ability to exploit natural variation , provides a variety of approaches to selectively manipulate the mouse genome and reveal the impact of genetic variants as well as discover missing heritability factors . to move the field forwards on a large scale , the international knockout mouse consortium ( ikmc ) will soon achieve its goal of having a mouse mutant or a targeted mesc for every gene , providing a crucial resource for functional annotation ( www.knockoutmouse.org ) . mouse disease clinics , which use broad - based phenotyping platforms , will assess these strains to understand gene function and model human disease ( http://eumodic.org ; http://nihroadmap.nih.gov/komp2/ ) . the complex trait community will soon generate up to 1000 strains that can be phenotyped for many traits and under many conditions ( http://www.complextrait.org/ ) . in support of these efforts , 45 of the most commonly used inbred strains that show a high degree of diversity are being analyzed for common variation or are being sequenced , providing powerful information on the origin and evolution of inbred strains of mice ( http://www.ensembl.org/mus_musculus/info/index ) . the challenge now is to expand our creativity by developing novel assays to detect a broad spectrum of phenotypes that are relevant to human health and disease . studies in mice and humans are thus poised to inform the other , resulting in new avenues for disease prevention , risk assessment , diagnosis and treatment . the future holds great promise as we follow the unwinding dna road towards a fuller understanding of the genetic basis for human health .