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 .
 the mouse is the leading organism for disease research . 
 a rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations . 
 however , one can also obtain desired gene mutations by using the following processes : targeted mutations that eliminate function in the whole organism or in a specific tissue ; forward genetic screens using chemicals or transposons ; or the introduction of exogenous transgenes as dnas , bacterial artificial chromosomes ( bacs ) or reporter constructs . 
 the mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation , and is therefore a powerful application for modeling human disease . 
 this poster review outlines the major genome manipulations available in the mouse that are used to understand human disease : natural variation , reverse genetics , forward genetics , transgenics and transposons . 
 each of these applications will be essential for understanding the diversity that is being discovered within the human population . 
Natural variation
Reverse genetics
Transgenics
Forward genetics
Transposons
Moving forward