copy number variation ( cnv ) is a structural genomic variation of the human genome that may either be inherited or caused by de novo mutation .
cnvs can range in size from kilobases ( kbs ) to several megabases ( mbs ) that have not been identified by conventional chromosomal analysis .
however , recent technology of genome - wide analysis such as comparative genomic hybridization ( cgh ) has led to the discovery of extensive genomic structural variation [ 13 ] .
a recent report using microarray technology revealed that as much as 12% of the human genome are variable in copy number .
these known cnvs are available from the interactive web - based database decipher ( database of chromosomal imbalance and phenotype in humans using ensembl resources , http://decipher.sanger.ac.uk/ ) .
the decipher database is a consortium comprised of an international network of more than 100 centers and has uploaded more than 2000 cases ( current statistics can be found on the decipher homepage ) .  
de novo mutations are more likely to contribute to the development of sporadic genomic disorders [ 6 , 7 ] . in psychiatric disorders , asd and schizophrenia ,
extension of genome - wide association studies ( gwas ) have led to the discovery of both inherited and de novo sporadic cnvs .
such cnvs resulted in altering gene dosage and dosage - sensitive gene expression , which may contribute to these disorders complexities .
these human genetics studies have detected several cnvs ( e.g. , 1q21 , 3q29 , 10q26 , 11p14 , 15q11 , 15q13 , 16p13 , 17p12 , and 22q11 ) .
this discovery suggests an important role for the strict regulation of gene dosage in asd and schizophrenia . to understand psychiatric disorders ,
while it is difficult to model human psychiatric phenotypes in animals ( e.g. , hallucinations and delusions characteristic of schizophrenia that are human specific ) , animal models may contribute to the elucidation of brain anatomy , behavioral characteristics , and molecular mechanisms that reflect aspects of human phenotypes .  
although there is a strong association between genetic rearrangement and psychiatric disorders ( e.g. , asd and schizophrenia ) , valid animal models that reflect etiology are rare .
several efforts have been made to generate mouse models of psychiatric disorders by conventional gene targeting , conditional gene targeting , and point mutation by chemical mutagens .
but these techniques are not enough to reflect complex human genomic rearrangements , such as large deletions , inversions , and duplications . in this regard ,
the cre / loxp - based chromosome engineering technique is useful to generate this kind of complex genomic rearrangements in the mouse genome . by using this chromosome engineering technique
, we can accomplish cnv - based unbiased animal models of psychiatric disorders . in this paper
, we focus on animal model of asd ( and schizophrenia ) which was generated by chromosome engineering , principle of this technology , and discuss for future directions .
genetic abnormalities such as point mutations , deletions , duplications , inversions , and translocations can be induced by exposure to x - ray radiation , chemical mutagens ( e.g. , n - ethyl - n - nitrosourea ( enu ) ) , conventional gene - targeting , or chromosome engineering .
the chemical mutagen , enu , induces single - base - pair substitutions in the genome causing mutations with partial functions [ 17 , 18 ] .
animal models containing genes with point mutations can be used to reveal the gene 's functional domain in vivo .
conventional gene targeting ( replacement ) is used to disrupt a gene ( inserting markers or reporters ) to determine a gene 's function .
conditional gene - targeting utilizing the cre / loxp and flp / frt system allows spatial and temporal control of gene expression .
chromosome engineering is based on cre / loxp technology , which can induce chromosome rearrangements ( deletions , duplications [ 10 , 20 , 21 ] , and inversions [ 22 , 23 ] ) in the mouse genome ( figure 1 ) .  
targeting vectors can be targeted in two orientations that result in deletion , duplication , or inversion .
each targeting vector has a loxp site and drug selection marker , neomycin resistance ( neo ) , or puromycin - resistant gene ( puro ) .
two loxp sites are sequentially inserted by each targeting vector into the mouse embryonic stem ( es ) cell genome .
the vectors are manipulated by hypoxanthine phosphoribosyl transferase ( hprt ) expression following cre recombinase expression in es cells .
clones which carry the desired chromosomal rearrangement are identified by various methods : drug selection by hypoxanthine - aminopterin - thymidine ( hat ) media , genomic southern blot analysis , fluorescent in situ hybridization ( fish ) , and cgh ( comparative genomic hybridization ) array .
although cgh array can not identify structural chromosome aberrations such as balanced reciprocal translocations and inversions , this technique is a powerful tool to detect cnvs from genome .   to inactivate a target gene or locus by chromosome engineering
the mutagenic insertion and chromosome engineering resource ( micer ) ( http://www.sanger.ac.uk/resources/mouse/micer/ ) was developed by dr .
allan bradley 's group , the wellcome trust sanger institute and is useful as a gene - targeting vectors resource .
these ready to use targeting vectors can be accessed through the ensembl mouse genome browser ( http://www.ensembl.org/index.html ) .
it is important to note that these targeting vectors use an insertion vector system rather than a replacement vector system .
given the same length of homologous sequence insertion vectors have a ninefold higher targeting efficiency than replacement vectors .
in spite of a strong association between asd ( and schizophrenia ) and cnv , animal models of cnv that reflect human genomic rearrangement are few .
these animal models were generated by chromosome engineering and have several psychotic phenotypes similar to those seen in patients with genomic rearrangement ( table 1 ) . in this section
we focus on 15q11 - 13 , 16p11.2 , and 22q11 locus , which are well - known copy number variant linked to asd ( or / and schizophrenia ) .
human chromosome region , 15q11 - 13 , is a complicated region that contains -aminobutyric acidreceptor a ( gabaa receptor ) clusters and several imprinting genes .
in addition to these genes , this locus includes noncoding small nucleolar rnas ( snornas ) that are located between snurf - snrpn and ube3a , which are paternally expressed and brain specific [ 27 , 28 ] .
prader - willi syndrome ( pws ) and angelman syndrome ( as ) are affected by changes in the 15q11 - 13 locus .
major clinical features of pws include low birth weight , short stature , small hands and feet , severe hypotonia , feeding difficulties , obesity associated with hyperphagia starting in early childhood , mild to moderate mental retardation , and learning and behavioral problems including obsessive - compulsive disorder and autism [ 29 , 30 ] . as patients exhibit developmental delay , gait ataxia , balance disorder , frequent laughter / smiling , easily excitable personality , hyperactivity , speech impairment , microcephaly , seizures , epilepsy , and abnormal eeg ( electroencephalogram ) . additionally , as patients often exhibit socialization and communication deficits , which are diagnostic criteria for asd [ 32 , 33 ] .
duplication of the 15q11 - 13 locus was first reported as a partial trisomy of chromosome 15 , and then two individuals with autistic disorder were reported .
this locus has been known as the most frequent cytogenetic abnormality in asd [ 36 , 37 ] .
generally patients with 15q11 - 13 duplication show hypotonia , delay in motor skills and language development , epilepsy , and cognitive and learning problems .
recently , michelson et al . reported a patient with severe intractable epilepsy who has familial partial trisomy 15q11 - 13 inherited from a mother who has schizophrenia .
autistic phenotype associated with 15q11 - 13 duplication , usually believed that maternal origin , ube3a is involved [ 3946 ] .
although maternal locus supposed to critical , paternally inherited patients had also developmental delay [ 44 , 4649 ] .
clinical reports have been accumulating but no mechanism has been addressed .   to address this question , nakatani et al .
this mouse was generated by chromosomal engineering based on the cre / loxp system , and it has a 6.3  mb duplicated locus in mouse chromosome 7c which is highly similar to human 15q11 - 13 ( figure 2(a ) ) .  
gene expression analysis revealed that paternally expressed genes , both ndn and snrpn , were twofold higher in paternally inherited mice ( patdp/+ ) than wild - type ( wt ) mice .
similarly , maternally expressed gene ube3a was twofold higher in maternally inherited mice ( matdp/+ ) than wt mice .
monoamine levels in patdp/+ adult mice , serotonin ( 5-ht ) , and their metabolites 5-hydroxyindoleacetic acid ( 5-hiaa ) were significantly downregulated in the midbrain and olfactory bulb .
also 5-ht content in developmental stage from postnatal 1 to 3 weeks in patdp/+ brain regions ( cortex , hippocampus , cerebellum , midbrain , hypothalamus , pons , and medulla ) was downregulated .
this indicates 5-ht signaling during the developmental stage was significantly impaired in the brains of patdp/+ mice .
5-ht influences not only mental condition ( mood , social behavior , appetite , aggression and sleep ) but also normal development of the central nervous system [ 5052 ] .
in addition to this , abnormal 5-ht levels have been found in asd patient blood cells .
treatment with serotonin reuptake inhibitors ( ssris ) have shown moderate success in recovering behaviors .  
behavioral tests revealed that patdp/+ mice display autistic behaviors such as less social interaction in the three - chamber social interaction test , abnormal ultrasonic vocalizations ( usvs )   in postnatal developing pups separated from their dams , and behavioral inflexibility in the morris water maze and barnes maze .
the phenotypes seen in patdp/+ mice indicate that these mice have impaired behaviors that include social interaction , communication , restricted interest , and resistance to change .
furthermore , patdp/+ mice showed anxiety - related phenotypes : decreased locomotor and exploratory activities in the open field and y - maze test , and long latencies in novelty suppressed feeding test .
these anxiety - related phenotypes frequently accompany autistic symptoms in humans [ 58 , 59 ] .
also the marble burying test , which is a useful test for the study of anxiety , obsessive - compulsive disorder ( ocd ) , and neophobia , found that the number of buried marbles was significantly low in patdp/+ mice .
deletion or duplication of the chromosome 16p11.2 locus was observed in nearly 1% of multiplex families with asd .
meta - analysis of patients with asd and/or developmental delay estimated that 16p11.2 locus deletion is associated with a 38.7-fold increase in the odds of asd / developmental delay .
on the other hand , 16p11.2 locus duplication is associated with a 20.7-fold increase in the odds of asd / developmental delay [ 6063 ] .
in addition to these , 16p11.2 deletion is associated with obesity , and duplication is associated with schizophrenia   as well as asd [ 60 , 66 ] .
notably , a brain anatomical abnormality ( abnormal head size ) has been reported to be associated with this locus .
for instance , patients with the 16p11.2 deletion had statistically significant macrocephaly and those with duplication had microcephaly .   a mouse model of human 16p11.2 deletion ( df/+ ) as well as duplication ( dp/+ ) has been reported   ( figure 2(b ) ) .
this locus includes 27 genes , spn , qprt , c16orf54 , kif22 , maz , prrt2 , c16orf53 , mvp , cdipt , sez6l2 , asphd1 , kctd13 , loc124446 , hirip3 , ccdc95 , doc2a , fam57b , aldoa , ppp4c , ypel3 , gdpd3 , mapk3 , and coro1a .  
young df/+ mice ( before weaning ) tend to be smaller than wt siblings , but as adults they are almost the same size as wt siblings and look healthy .
interestingly , 16p11.2 cnv mice , df/+ and dp/+ mice have opposite phenotypes . in a novel environmental cage
, df/+ mice displayed longer distance traveled and time spent walking as compared with wt mice .
in contrast , dp/+ mice traveled a shorter distance and spent less time walking as compared with wt mice . additionally , df/+ mice were significantly active in both dark and light period .
these results indicate that 16p11.2 locus affects not only physical activity but also diurnal activity and sleeping related symptoms .
also , a brain anatomical study using magnetic resonance imaging ( mri ) identified several regional changes in 16p11.2 cnv mice .
for instance , df/+ mice showed increased volume of several brain regions ( percentage of total brain volume ) : forebrain , superior colliculus , fornix , hypothalamus , mammillothalamic tract , medial septum , midbrain , and periaqueductal grey .
these brain regional volumetric changes were more significant between df/+ and dp/+ than betweendf/+ and wt mice .  
microdeletion of chromosome 22q11 is found in 1 out of every 4000 live births , making it one of the most common interstitial deletions .
this 22q11.2 microdeletion causes craniofacial , cardiovascular abnormalities , immunodeficiency , hypocalcaemia , short stature , and cognitive dysfunctions [ 6971 ] .
microdeletion of this region accounts for 1 - 2% of the cases of people with schizophrenia [ 72 , 73 ] .
also , this locus accounts for up to 1 - 2% of cases of sporadic schizophrenia [ 7476 ] .
volumetric reduction in total brain volume includes cortical regions ( e.g. , frontal , parietal , temporal , and occipital lobes ) , hippocampus , and cerebellum [ 7786 ] .
however , inconsistency in these neuroimaging reports may be due to the small numbers of subjects used and differences in methodology .
yet these neuroanatomical reports are informative because some abnormalities are consistent with phenotypes of those who have non-22q11.2 ds - associated schizophrenia .
the majority of deletions in this locus are 3  mb deletions ( 90% of the cases ) , but 1.5  mb deletions ( < 10% of the cases ) contain 28 known genes which include critical genes and increased risk of mental disorders [ 73 , 87 ] .   
animal models of the human 22q11.2 deletion were generated by 2 groups , and both groups used chromosome engineering [ 14 , 88 ] ( figure 2(c ) ) .
these mouse models , df(16)a   and lgdel/+ , include 1.5  mb critical regions , and both of them display several behavioral abnormalities , such as deficits in working memory , sensorimotor gating , and fear conditioning [ 14 , 8992 ] .
working memory deficits are becoming one of the main features of patients with schizophrenia , thus these animal models are supposed to reflect some aspects of 22q11.2 ds syndrome phenotype .
in addition to behavioral abnormalities in this mouse , diminished 22q11 locus dosage disrupts cortical neurogenesis , interneuron migration , dendritic complexity , and formation of excitatory synapses .
although , several interesting phenotypes have been reported in this mutant mouse , there are no studies published about brain structural abnormalities even though several brain abnormalities have reported in human studies .
these brain anatomical changes and molecular mechanisms that underlie these phenotypes will be interesting to elucidate and will be addressed by using brain imaging techniques .
application of new technologies , such as comparative genomic hybridization ( cgh ) and next - generation sequencing , will reveal more additional genomic rearrangements related to psychiatric disorders .
thus , to analyze both phenotypes and underlying molecular mechanisms that originate from genetic rearrangements , animal models will be a powerful tool . in this context
reported that they generated several hundred mice and embryos which have one loxp and lacz site at a random genomic positions that inserted by sleeping beauty - based transposition system .
these lines are mapped in transposon and recombinase associated chromosomal engineering resource database ( tracer , http://tracerdatabase.embl.de/fmi/iwp/res/iwp_home.html ) .
it is an orthodox approach to narrow down the region by systematically insertion of loxp combining the existed lines such as above tracer .
generating bacterial artificial chromosome ( bac ) transgenic mice is another way to identify critical genes .
bac transgenes inserted to the genome faithfully recapitulate chromosomal endogenous gene expression , since bac transgenic mice may appropriate animal model of gene duplication .
also transient overexpressing ( or knockdown ) each transcript in developmental brain is possible strategy .
identified a responsible gene kctd13 in 16p11.2 locus which causes brain malformation by using zebrafish .
use of these technologies in generating valid and etiology - based animal model of psychiatric disorders will contribute to the development of drugs against disorders and elucidation of molecular mechanisms that underlie these psychiatric disorders .
 the development of genetic technologies has led to the identification of several copy number variations ( cnvs ) in the human genome . 
 genome rearrangements affect dosage - sensitive gene expression in normal brain development . 
 there is strong evidence associating human psychiatric disorders , especially autism spectrum disorders ( asds ) and schizophrenia to genetic risk factors and accumulated cnv risk loci . 
 deletions in 1q21 , 3q29 , 15q13 , 17p12 , and 22q11 , as well as duplications in 16p11 , 16p13 , and 15q11 - 13 have been reported as recurrent cnvs in asd and/or schizophrenia . 
 chromosome engineering can be a useful technology to reflect human diseases in animal models , especially cnv - based psychiatric disorders . 
 this system , based on the cre / loxp strategy , uses large chromosome rearrangement such as deletion , duplication , inversion , and translocation . 
 although it is hard to reflect human pathophysiology in animal models , some aspects of molecular pathways , brain anatomy , cognitive , and behavioral phenotypes can be addressed . 
 some groups have created animal models of psychiatric disorders , asd , and schizophrenia , which are based on human cnv . 
 these mouse models display some brain anatomical and behavioral abnormalities , providing insight into human neuropsychiatric disorders that will contribute to novel drug screening for these devastating disorders . 
1. Introduction
2. Chromosome Engineering in Mice
3. Animal Models Based on Human CNVs
4. Future Perspectives