Artificial Chromosome Based Trans Genes in Genome Function Studies

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Articial chromosome-based transgenes in the study of genomefunction

Jason D. Heaney, Sarah K. BronsonDepartment of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine,The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033-0850, USA

Received: 7 February 2006 / Accepted: 6 April 2006

Abstract

The transfer of large DNA fragments to the mouse

genome in the form of bacterial, yeast or phageartificial chromosomes is an important process inthe definition of transcription units, the modeling ofinherited disease states, the dissection of candidateregions identified by linkage analysis and the con-struction of in vivo reporter genes. However, as withsmall recombinant transgenes, the transferred se-quences are usually integrated randomly often withaccompanying genomic alterations and variableexpression of the introduced genes due to the site ofintegration and/or copy number. Therefore, alterna-tive methods of integrating large genomic transgenesinto the genome have been developed to avoid thevariables associated with random integration. Thisreview encourages the reader to imagine the largevariety of applications where artificial chromosometransgenes can facilitate in vivo and ex vivo studiesin the mouse and provides a context for makingthe necessary decisions regarding the specifics ofexperimental design.

Characteristics and Applications of Articial

Chromosome Transgenes General characteristics of articial chromo-somes. The analysis of large, complex genomes re-quires various tools for the mapping and cloning ofDNA. While plasmid- and cosmid-based cloning canbe useful for the identification of individual genes,their limited insert size (20 40 kb) makes it difficultto produce large-scale physical maps that order DNA

over mega-base distances. The development of yeastartificial chromosomes (YACs) was the first advancein cloning technology that allowed for detailed

physical mapping of large segments of mammalianchromsomes (Figure 1a) (Burke et al. 1987; Imai andOlson 1990; Silverman et al. 1991). YACs provide thelargest insert capacity of all artificial chromosomes,and while they are capable of maintaining genomicinserts >1 Mb in size, the average size of most YACclones lies within the 400 600 kb range (Burke et al.1987). Although their large insert capacity makesYACs seem ideal for transgenic applications, severaltechnical issues must be considered when workingwith this cloning system. Because YACs are main-tained as linear molecules, they are prone to shearingby mechanical stresses. YACs can also be difficult topurify from similarly sized endogenous yeast chro-mosomes as they are typically separated fromendogenous chromosomes by pulse field gel electro-phoresis (PFGE) of agarose-embedded yeast cells.Furthermore, YAC libraries have a high rate of: 1)insert chimerism, i.e. co-cloning of non-contiguoussequences in a single clone, and 2) insert rearrange-ments/deletions, a result of the active homologousrecombination machinery of the yeast host (Kouprinaet al. 1994; Larionov et al. 1994; Neil et al. 1990).Both of these events can lead to conflicting sequenceinformation when compared to existing databases.

The efficient recombination machinery of the yeasthost does, however, provide a mechanism by whichmarkers and modifications can be inserted into thegenomic clone for functional studies of YACs in vivo(Campbell et al. 1991; Reeves et al. 1990).

Because of the inherently unstable nature ofYACs, alternative cloning systems able to propagatelarge genomic fragments were sought. The first YACalternative developed was the P1 phage cloningsystem (Glover, D. M. and others 1995; Sternberg1990). The P1 cloning system has many advantagesCorrespondence to: Sarah K. Bronson; E-mail: [email protected]

DOI: 10.1007/s00335-006-0023-9 Volume 17, 791 807 (2006) Springer Science+Business Media, Inc. 2006 791

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over YACs. The bacterial host is recA ) ,and thereforedoes not readily rearrange the genomic insert byhomologous recombination (Sternberg 1990). Addi-tionally, the P1 clones are maintained as a singlecopy (Sternberg 1990). Previous studies have dem-onstrated that genomic DNA propagated in high-copy plasmids is prone to deletions and rearrange-ments in the bacterial host, suggesting the single

copy nature of P1 clones further contributes to theirstability (Kim et al. 1992). Additionally, the super-coiled, circular nature of P1 clones makes them lesssusceptible to mechanical shearing. While P1 phageclones are much more stable than YACs, theirgenomic insert size is severely limited. The P1 phagehead can accommodate up to only 110 kb of DNA(Giraldo and Montoliu 2001; Glover, D. M. andothers 1995).

Although the stability of P1 phage clones makesthem an attractive alternative to YACs, additionalcloning systems that are just as stable, but not as

restricted in genomic insert size or dependent on anelaborate in vitro packaging system, were desired.The bacterial artificial chromosome (BAC) cloningsystem is based on the Escherichia coli (E. coli ) fer-tility (F) factor (Shizuya et al. 1992). It was previouslydemonstrated that F plasmids are able to maintainbacterial DNA inserts as large as 1 Mb; however, themaximum observed mammalian genomic insert size

is 300 kb (Kim et al. 1996; Shizuya et al. 1992).Various BAC vector backbones exist with some dif-ferences in sequence content; however, they are allbased on the pBAC108L vector (Figure 1b) (Frengenet al. 1999; Kim et al. 1996; Shizuya et al. 1992; Zenget al. 2001). BACs are maintained in one or twocopies in the bacterial host by a trio of F factor genes( parA, parB, parC ), and all BAC vectors containeither a single wild-type loxP site or, in someinstances, an additional mutant loxP511 site (Moriet al. 1986; Shizuya et al. 1992). As with the P1 phagesystem, BACs are circular molecules resistant to

ARS1 CEN4TRP1 URA3Genomic Insert (0.2 2 Mb)

Telomeres

pBeloBAC11

lox P CMR

OriS

RepE

parA

Hind III Bam HI

lacZ

parC

parB

cosN

SacBII

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Kan R / TN903

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lox P

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Bam HI,Eco RI

Bam HI,Eco RI

pCYPAC2

ARS1 CEN4TRP1 URA3Genomic Insert (0.2 2 Mb)ARS1 CEN4TRP1 URA3Genomic Insert (0.2 2 Mb)

Disrupted SUP4

pBeloBAC11

lox P R

OriS

E

parA

parC

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a

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Kan R / TN903

P1 PlasmidReplicon

lox P

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Bam HI,Eco RI

Bam HI,Eco RI

pCYPAC2

c

Fig. 1. Common artificial chromosome cloning systems ( a) A generic YAC clone. Ligation of a genomic fragment into theYAC arms disrupts SUP4 and releases ochre suppression, which in Ade2 -ochre hosts disrupts adenine metabolism andresults in the accumulation of a red pre-metabolite. Sequences necessary for centromere function (Cen4), autonomousreplication (ARS1), and selection in the yeast host (URA3 & TRP1) are indicated. ( b) BAC vector pBeloBAC11 is a modifiedversion of pBAC108L, which contains the LacZ (b-galactosidase) gene at the genomic DNA cloning site. Bacteria har-boring a BAC with a genomic insert are white in the presence of X-gal; bacteria harboring just the vector are blue in thepresence of X-gal due to the activity of LacZ . All BAC vectors contain a wild-type lox P site. ( c) The original PAC vectorpCYPAC2, which contains a stuffer (pUC-LINK) at the Bam HI / Eco RI cloning sites, separating SacBII from its promoter.pCYPAC2 contains both the P1 phage lytic and plasmid replicons. Disruption of SacBII expression by a cloned genomicinsert allows for positive selection in sucrose. All PAC vectors contain a wild-type lox P site. Figures are not to scale.

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mechanical shearing and can be isolated using con-ventional plasmid purification protocols (Yang et al.1997). Just as with P1 phage clones, BACs aremaintained in a recA- host and are much more stablethan YACs.

Phage artificial chromosomes (PACs) weredeveloped to combine the unique features of BACsand P1 phage (Figure 1c) (Frengen et al. 2000; Ioan-nou et al. 1994). The PAC is a modified version ofthe P1 phage plasmid that, after ligation to partiallydigested genomic DNA, is electroporated as a cir-cular molecule into the bacterial host, rather thanbeing packaged into bacteriophage particles. Byaverting the packaging step, PAC clones can bemaintained as genomic inserts of the same size asBACs (Ioannou et al. 1994). Additionally, somePACs contain the P1 lytic replicon, which can beactivated by IPTG to increase PAC copy number

prior to DNA purification, resulting in increasedDNA yield (Ioannou et al. 1994). The stability andrelatively large insert capacity of BAC and PACclones have made them ideal for high-resolutionphysical mapping. As such, BACs and PACs werethe cloning system of choice for constructing phys-ical maps of the publicly-funded human and mousegenome sequencing projects. With the current se-quence information available for most BAC and PACclones, these artificial chromosomes have become apopular resource for artificial chromosome-basedtransgenes.

Arti cial chromosome transgenes and the study of gene function and regulation. During the earlyto mid 1990s, several groups first described the useof YACs, BACs, and PACs as transgenic vectors formice (Choi et al. 1993; Jakobovits et al. 1993; Lambet al. 1993; McCormick et al. 1996; Morimoto et al.2002; Radomska et al. 2002; Schedl et al. 1992;Schedl et al. 1993a; Schedl et al. 1993b; Smith et al.1995; Strauss et al. 1993; Yang et al. 1997). Thesegenomic transgenes have become an invaluable toolfor the modeling of inherited disease states, defin-ing transcription units, and dissecting candidate

regions identified by linkage analysis. Many largegenes, such as hypoxanthine phosphoribosyl trans-ferase ( Hprt ), amyloid precursor protein ( App ), andClock , as well as gene complexes, such as the b- globin locus, the immunoglobulin loci, and themajor histocompatibility complex, cannot be effec-tively transferred in their entirety to the mousegenome without the use of artificial chromosomes(Antoch et al. 1997; Bronson et al. 1991; Lamb et al.1993; Lawrance et al. 1987; Mendez et al. 1997;Porcu et al. 1997; Scheerer and Adair 1994; Smith etal. 1997). Additionally, a significant number of both

large and small genes do not reproduce endogenousexpression patterns in a transgenic setting, likelydue to the exclusion of distant regulatory sequencesfrom the transgenic construct, and recombinantcDNA-based constructs may omit critical intronicregulatory sequences (Giraldo and Montoliu 2001;Jaenisch 1988).

For many transgenic mouse models of humandisease, proper spatial and temporal expression ofthe transgene is essential to accurately model thedisease state and determine the role of the intro-duced sequences in disease progression. For example,cDNA transgenes of wild-type and mutant deriva-tives of APP have been used to generate mousemodels of Alzheimers disease through the overpro-duction of b-amyloid protein, the amyloidogenicpeptide derivative of APP (Selkoe 1994). Unfortu-nately, mouse models utilizing APP cDNA transg-

enes display very low and/or inappropriatelyregulated transgene expression, and in several in-stances, the principle features of Alzheimers diseaseare not reproduced (Games et al. 1995; Hsiao et al.1996; Kammesheidt et al. 1992; Kawabata et al.1991; Nalbantoglu et al. 1997; Quon et al. 1991;Wirak et al. 1991). However, in YAC transgenic miceharboring normal or mutant forms of APP , temporaland spatial expression of APP and its derivativesreflects that of the endogenous mouse locus andyields a superior animal model of the human disease(Chiocco et al. 2004; Lamb et al. 1993; Lamb et al.1997).

Artificial chromosome-based transgenes havealso proven to be important tools for the study ofdiseases associated with cytogenetic abnormalities.YACs and BACs have been used to generate panels ofmice transgenic for regions of human chromosome21 (HSA21). Such panels allow the identification ofgenes that when triplicated, contribute to Downsyndrome (Smith et al. 1995; Smith et al. 1997).While these mice could be generated using cDNAtransgenes derived from known genes located onHSA21, the large size of YAC and BAC transgenesallows for long-range screening of HSA21 for genes

associated with Down syndrome phenotypes. It isalso likely that many of the observed phenotypesinvolve contiguous stretches of the chromosomeencompassing many genes, which would be difficultto reproduce with cDNA transgenes. In addition,expression of genes contained on a YAC or BACtransgene should more closely reflect that of thenative locus due to the near-endogenous chromatinenvironment of the introduced sequences. When thetemporal and tissue-specific expression of a gene isincompletely understood, choosing to use a heter-ologous promoter to control expression of a cDNA

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transgene is risky, and may not properly reproducethe desired phenotype.

Genomic transgenes are also an important re-source for in vivo studies of gene regulation. Manyfunctional assays of promoter/enhancer functionhave been studied by linking putative regulatory re-gions to reporter constructs, such as luciferase, intransient transfection or transgenic settings. Unfor-tunately, these recombinant constructs may notaccurately reproduce the transcriptional regulationmediated by the native chromatin environment. Inan example of the utility of YACs and BACs for invivo gene regulation studies, the regulatory regionsof the muscle-specific transcription factors Myf5 andMrf4 were delineated with BAC transgenes . In theseexperiments, a panel of transgenic mice was gener-ated using a series of BACs containing the Myf5/ Mrf4 locus and varying amounts of the 5 flanking

region (Carvajal et al. 2001). By introducing differentreporter constructs into the Myf5/Mrf4 locus of theBAC and using the modified BACs as transgenes, theauthors were able to identify regulatory regions inthe 5 flanking region necessary to drive appropriatespatial and temporal Myf5 and Mrf4 expression invivo . While some of these regions had been previ-ously characterized using small recombinanttransgenes, the authors were able to identify addi-tional regulatory elements located in the distal 5 region of the Myf5/Mrf4 locus (Carvajal et al. 2001).

In addition to analyzing the function and regu-lation of known genes, YAC and BAC transgenes areimportant resources for the complementation of ge-netic linkage groups associated with developmentaldefects. The detailed physical maps with sequenceinformation that depended so heavily on theemerging technologies of artificial chromosomes arenow allowing rapid identification of candidate genesfor single gene and QTL effects. In many instancesdefinition of the causative gene or contributing QTLcan be accomplished by complementation studies.Again, as with the Down syndrome studies, moresequence can be initially covered by transferringartificial chromosomes to a mutant cell or animal,

and the near endogenous expression of the transgeneis more likely to complement the phenotype. Schedland colleagues were the first to demonstrate thatartificial chromosomes could complement a mutantphenotype in mice (Schedl et al. 1993b). A YACtransgene containing the tyrosinase locus wasutilized to correct the albino phenotype of micecontaining the naturally occurring tyrosinase genemutation ( cc genotype). While minigenes of tyrosi-nase were inconsistent in their ability to comple-ment the albino phenotype, the YAC transgenecompletely rescued the mutant phenotype due to the

presence of distant regulatory regions absent fromthe minigenes.

With the advent of efficient methods for modi-fying BACs in their bacterial host, BAC transgeneshave become reporters for the molecular identifica-tion of cell and tissue types in vitro and in vivo

(Gong et al. 2002; Gong et al. 2003; Lee et al. 2001;Muyrers et al. 1999). BACs can be modified to con-tain a reporter (e.g., GFP, luciferase, b-galactosidase,alkaline phosphatase) whose expression is regulatedby the promoter/enhancer sequences of a locuscontained within the BACs. These BAC reportertransgenes can then be used to track the influence ofenvironmental signals, or the effect of geneticalterations in cis or trans, on gene expression.A modified BAC transgene could be utilized to trackthe functional differentiation of cells in vitro or post-transplantation of stem or progenitor cells in vivo .

BACs could be modified to express a drug resistancegene that would allow enrichment of, or selectionfor, a particular cell type. While small recombinanttransgenes can be used in a similar manner, the lowdegree of tissue-specificity attributed to minimalpromoters, particularly after random integration intothe genome, is generally not adequate to accuratelyidentify highly specialized cell types. Unfortunately,the complications of random integration (positioneffect, variable copy number, and insertionalmutagenesis of the host genome) extend to artificialchromosome-based transgenes and must be consid-ered during experimental design.

Random Integration of Arti cial Chromosome Transgenes

Stable vs. variegated position effects. It is welldocumented that endogenous sequences surroundingthe site of random integration can alter expectedtransgene expression levels and patterns (position-dependent expression) (Caron et al. 2002; Giraldo andMontoliu 2001; Hatada et al. 1999). The mechanismsunderlying position effects have been subcategorizedinto two groups: stable position effects and silencing

position effects. Stable position effects are pancellu-lar and result in differences in expression between atransgene and the endogenous locus or similartransgenes integrated into different genomic sites.Transgene expression levels are influenced eitherpositively or negatively by the regulatory elements atthe site of integration. Stable position effects aregenerally associated with small recombinanttransgenes that contain minimal promoter se-quences, rendering them unable to override the ef-fects of the promoters and enhancers at the site ofintegration.



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The second type of position effect, silencing po-sition effect, which is also referred to as positioneffect variegation (PEV), is characterized by an epi-genetically inherited silencing of gene expression ina clonal subpopulation of cells (Karpen 1994). How-ever, in cells that do express the transgene, expres-sion levels are at or near those of the endogenousgene (Alami et al. 2000; Festenstein et al. 1996; Milotet al. 1996; Robertson et al. 1995). In PEV, hetero-cellular transgene expression is the direct result ofrandom heterochromatinization of euchromaticDNA juxtaposed with heterochromatin (Alami et al.2000; Festenstein et al. 1996; Milot et al. 1996;Robertson et al. 1995). This phenomenon was firstdescribed in Drosophila where the white gene(responsible for red eye color) translocated into aheterochromatic region of DNA (Dillon and Festen-stein 2002). The subsequent silencing of the white

gene in a subpopulation of cells resulted in a mosaicexpression that gave rise to red and white patches inthe mature Drosophila eye (Muller 1930). Thisphenomenon has also been described in mammaliancells; and it has been demonstrated that the orien-tation of the transgene with respect to the sur-rounding chromatin can influence silencing (Fenget al. 2001). In tissues where a straightforward meansof observing PEV does not exist, silencing positioneffects will present as a decrease in total tissueexpression, and may be mistaken for a stable posi-tion effect. Only careful analysis of transgeneexpression within individual cells can identify PEV.

In most instances, the introduction of largetransgenes (>40 kb) into the genome of embryonicstem cells (ES cells) or fertilized eggs has also reliedon random integration. Interestingly, it has beenobserved that expression from artificial chromo-some-based transgenes is less likely to be subject tostable and silencing position effects than smallerrecombinant transgenes (Giraldo and Montoliu2001). Presumably, inclusion of native regulatoryelements within the YAC or BAC transgene over-rides the influence of regulatory elements located atthe site of random integration. Additionally, inclu-

sion of endogenous insulator elements within thelarge construct may help to maintain proper epige-netic organization of the transgenic sequence, thusdecreasing the possibility of position effect variega-tion. While the regulatory elements necessary forproviding position-independent transgene expressionhave not been identified for many genes, thosenecessary for position-independent expression of theT-cell surface antigen CD2 and the genes within theb-globin locus have been well characterized (Alamiet al. 2000; Bungert et al. 1995; Festenstein et al.1996; Lang et al. 1988; Li et al. 2000; Porcu et al.

1997; Tanimoto et al. 1999). These studies led to thedefinition of the locus control region (LCR), a cis-acting element required for transgene expressionapproximately proportional to the number of copiesintegrated into the mouse genome (Alami et al.2000). LCRs, which are marked by DNase I hyper-sensitivity sites, contain strong enhancers of tran-scription, responsible for maintaining an openchromatin structure within both the endogenouslocus and transgenes at the appropriate stage ofdevelopment, as well as within appropriate tissues(Alami et al. 2000; Behringer et al. 1990; Bungertet al. 1995; Festenstein et al. 1996; Forrester et al.1987; Fournier and Ruddle 1977; Li et al. 2000; Porcuet al. 1997; Tanimoto et al. 1999; Tuan et al. 1985).

It would seem that the selection of YAC or BACclones containing all the regulatory elements of agene would eliminate position effects. While most

studies suggest that LCRs are indeed capable ofsuppressing stable and silencing position effects,there is growing evidence that even an intact LCR ona YAC transgene does not always protect againstPEV (Alami et al. 2000; Li et al. 2000; Porcu et al.1997). In these experiments, YAC transgenes of theb-globin and the mouse Ig j light chain locus wereshown to be subjected to silencing position effects insome founder lines even though they contained anintact LCR and were not pericentromerically inte-grated. It is possible these transgenes integrated intoan area of constitutive heterochromatin found out-side the centromeric regions and that the LCR wasnot capable of suppressing PEV (Alami et al. 2000). Inaddition, the location of an LCR has not been iden-tified, or even demonstrated to exist, for many locimaking it difficult to identify YAC or BAC clonesthat will be position-independent. It is possible thatother insulator elements, such as nuclear scaffold ormatrix attachment regions (S/MARs), which do nothave a transcriptional enhancement function, areresponsible for defining regions of euchromatin formany loci (Gerasimova and Corces 2001; Laemmliet al. 1992; Stief et al. 1989).

Copy number-dependent expression and repeat-based gene silencing. Transgenes typicallyintegrate into the mouse genome as head-to-tailconcatamers at a single genomic locus (Costantiniand Lacy 1981; Jaenisch 1988). Very rarely, two sitesof integration have been identified in founder mice(Jaenisch 1988). While it is common for 50 or morecopies of a small recombinant transgene to integrateinto the genome, YAC and BAC transgenic miceusually do not contain more than five copies, al-though rare transgenic founders with more than fivecopies have been identified (Antoch et al. 1997;



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Hodgson et al. 1996; Li et al. 2000; Schedl et al.1993b; Smith et al. 1997). Interestingly, it has alsobeen shown that individual copies of a YAC or BACtransgene within a single founder mouse may vary insequence content due to deletions or rearrangementsthat occurred prior to or during integration (Kaufmanet al. 1999; Peterson et al. 1998). The failure to detectsuch rearrangements or deletions may result in aloss of transgene expression due to: 1) a loss of po-sition-independence of the transgene and/or genesilencing at the locus due to the lack of a copy of aYAC that contains an intact regulatory region, or 2) agene within the construct appearing to be intact,when in reality, its content is split between twodifferent copies of the insert.

Intuitively, more copies of a transgene integratedinto the mouse genome should lead to higher geneexpression. However, there is usually not a linear

relationship between copy number and expressionlevel when different transgenic lines containing thesame transgene are compared. In addition to positioneffects, it has been observed that, in some instances,multiple tandem repeats of recombinant transgenescan lead to silencing of transgene expression (Gar-rick et al. 1998; Henikoff 1998). This phenomenon isoften observed with repeats of transgenes containingprokaryotic sequences, such as the LacZ gene, whichare CpG-rich and prone to methylation, or with re-peats of transgenes that have been condensed intoheterochromatin (Chada et al. 1985; Dillon andFestenstein 2002; Guy et al. 1996; Guy et al. 1997;Henikoff 1998). While methylation can, in rare in-stances, directly block transcription factor binding torepress transcription, there is much more evidenceto suggest that methylation of DNA and introduc-tion of heterochromatin are intimately linked duringgene silencing (Li 2002). It has been demonstratedthat methylated DNA recruits transcriptionalrepressors, such as MeCP2, which associate withco-repressors containing histone deacetylase activityto induce heterochromatin formation (Li 2002; Nanet al. 1998). Extending these results to transgenes,the sequence content of tandem repeats and the

tendency of those repeats to be methylated couldstimulate transgene silencing through heterochro-matin formation. It can be postulated that, with anincrease in transgene copy number and potentialmethylation sites for repressor binding, there is anincreased propensity for transgene silencing throughthe recruitment of HDAC activity to the site oftransgene integration.

For those YAC or BAC transgenes containing theelements necessary to confer position-independentexpression, a linear relationship between copynumber and gene expression is generally observed (Li

et al. 2000; Peterson et al. 1998). However, in rareinstances where many (eight or more) copies of alarge transgene integrate into the genome, the linearrelationship between copy number and expressionlevel is lost, suggesting that even large transgenicconstructs are prone to repeat-induced gene silenc-ing (Henikoff 1998; Li et al. 2000). As with smallertransgenes, it is likely that the repetitive sequencecontent of a large genomic transgene will influencesusceptibility to silencing.

Insertional mutagenesis. A less common phe-nomenon observed during random transgene inser-tion into the mouse genome is the generation ofphenotypes caused by the integration event itself.During random integration, it is possible for atransgene to insert into either the coding or theregulatory sequences of an endogenous gene, result-

ing in the disruption or alteration of gene expression(Krulewski et al. 1989; Miao et al. 1994; Rachel et al.2002; Ross et al. 1998). In most instances, insertionalmutagenesis will not result in an unexpected phe-notype because only one allele is mutated during theintegration event. However, if mutation of one alleleresults in haploinsufficiency, or if the transgene isbred to homozygosity, a phenotype resulting fromthe disruption of an endogenous gene may be ob-served. More recently it has been demonstrated thattransgene insertion may also have long range effectson endogenous gene expression. A recent study inhamster cells demonstrated that transgene insertioncan alter methylation and expression patterns overvast regions of the genome (Muller et al. 2001). Thiseffect is most likely linked to the site of integrationand interactions with distant chromosomal regionsfollowing chromatin folding.

Random integration and the interpretation of transgene function. For most experiments utilizingYACs or BACs as transgenes, expression levels andpatterns that mimic the endogenous gene are de-sired. For example, when comparing the function ofdifferent YAC or BAC transgenes in complementa-

tion of phenotype experiments, variability in, orsilencing of, transgene expression due to the site ofintegration can mask the ability of a transgene torescue the mutant phenotype. Similarly, BAC-basedreporter transgenes are only valuable if the expres-sion of the transferred gene mimics the endogenouslocus. For experiments utilizing multiple BACs,each will have integrated into different locus andsubtle influences conferred by these loci on trans-gene expression may further complicate any directcomparisons of function. Additionally, consideringthe variability in the number of integrated copies,



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the potential for repeat-induced silencing and diffi-culties in discerning insert integrity, it can clearly beseen why multiple founder lines must be carefullyscreened for proper levels of transgene expressioneven with YAC or BAC transgenes that are expectedto be position-independent. Finally, undesiredmutations of endogenous genes are of particularconcern when studying complementation of pheno-type. Phenotypes resulting from insertional muta-genesis and not the transgene itself can complicatethe interpretation of any functional role attributed tothe introduced sequences. In addition, insertionalmutagenesis can result in a misinterpretation of theeffect of gene overexpression in transgenic mice, orthe effect of a mutation introduced into a YAC orBAC transgene on gene function.

Various approaches have been employed tominimize the variables of random integration when

working with both large and small transgenes. Thesealternatives rely on either maintaining the trans-genic sequence as a freely segregating chromosomeor the precise recombination of the transgenic se-quence into a pre-selected site within the mousegenome. Both strategies minimize or eliminate po-sition effects, abolish copy number variability, andin most instances disallow insertional mutagenesisof the host genome.

Alternatives to Random Integration

Transchromosomes. If transgenic sequences couldbe introduced into the mouse genome as freely seg-regating chromosomes, the complications associatedwith random integration would be avoided. To thisend, two groups have utilized microcell-mediatedchromosome transfer to introduce large fragments ofhuman chromosomes into the mouse genome(Fournier and Ruddle 1977; Hernandez et al. 1999;Oberdoerffer et al. 2005; Tomizuka et al. 1997;Tomizuka et al. 2000). In this approach, irradiatedmicrocells are generated from human or human-mouse hybrid cell lines containing human chromo-somes randomly or site-specifically tagged with the

bacterial aminoglycoside 3 -phosphorylase gene ( neo )to provide resistance to the antibiotic G418. Themicrocells, each containing a fragment of a humanchromosome, are then fused to embryonic stem (ES)cells, introducing the human sequence into themouse genome (Hernandez et al. 1999; Tomizukaet al. 1997; Tomizuka et al. 2000). Chromosomalfragments containing a centromere are maintainedas freely segregating transchromosomes in themicrocell-hybrid ES cell (Hernandez et al. 1999;Tomizuka et al. 1997; Tomizuka et al. 2000). Whilethis method of introducing foreign DNA into the

mouse genome is more difficult technically thanthose described previously, it does provide severaladvantages. Transchromosomes are very large(50 kb 4 Mb), even when compared to YAC or BACtransgenes (100 kb 2 Mb). The large size of thetranschromosomes can be used for the generation ofmouse models for human diseases resulting fromduplications or deletions of large segments of achromosome, such as Down syndrome, or human-izing mice to produce human antibodies for thera-peutic purposes. To some extent these experimentshave been accomplished with YAC or BAC transg-enes; however, with the multi-megabase size ofsome loci, such as those of the immunoglobulinchains, it is impossible to transfer the entire regioninto the mouse genome using these cloning systems(Choi et al. 1993; Mendez et al. 1995; Mendez et al.1997). In addition to their large size, the ability of

transchromosomes to be maintained independentlyeliminates all of the variables observed with randomintegration.

Although transchromosomes seem to provide anideal method for generating transgenic mice fromlarge constructs, there are problems that make thisapproach less desirable. Under nonselective condi-tions, transchromosomes were lost in 0.1 % to 3.2 %of ES cells per population doubling, suggesting thattranschromosomes segregated properly during mito-sis in only a subpopulation of cells (Tomizuka et al.1997; Tomizuka et al. 2000). As expected, with theinstability of transchromosomes in the ES cell gen-ome, only a subset (30 80% ) of chimeric mice gen-erated from transchromosomal ES cells containedhuman sequences (Hernandez et al. 1999; Tomizukaet al. 1997; Tomizuka et al. 2000). In addition,transchromosomes did not pass efficiently throughthe germline of chimeric mice, with 0 % to 40 % ofchimera progeny carrying the transchromosome.Furthermore, retention of transchromosomes in cellpopulations within transgenic mice was variable,with only 65 % to 91 % of cells within a transgenicmouse carrying the transchromosome (Tomizuka etal. 1997; Tomizuka et al. 2000). This resulted in a

heterocellular transgene expression pattern similarto what is observed during transgene silencing fol-lowing random integration. With a variable numberof cells expressing the transgenic sequences, it couldbe difficult to determine the effect of a transchro-mosome when used in either an overexpressionmodel or complementation of phenotype study.

Cre recombinase-mediated loxP recombination into the ES cell genome. Cre recombinase-mediated loxP recombination has been an important tool inthe advancement of knockout mouse technology.



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The bacteriophage P1 Cre recombinase mediatesintra- and inter-molecular site-specific recombina-tion between loxP sites (Hoess et al. 1982). Under theregulation of different promoter sequences, Creexpression can be directed to a variety of cell types atdifferent stages of development. While the Cre/ loxP

system was initially used in mice to remove endog-enous sequences from the mouse genome, it becameapparent this technology could also be utilized tointroduce a single copy of a transgene into the mousegenome by precise recombination events. For suchrecombination events to occur, loxP sites must beintroduced into the mouse genome by homologousrecombination or random integration and a selectionscheme must be employed to identify the desiredrecombination event(s).

One method of introducing transgenes into theES cell genome by Cre/ loxP recombination has been

termed recombinase-mediated cassette exchange(RMCE) (Feng et al. 1999; Soukharev et al. 1999). Inthis system, a loxP cassette is introduced into the EScell genome at a pre-selected locus or by randomintegration to generate a loxP target site in the hostgenome. In general, these constructs employ two loxP sites, in opposite orientation, flanking a posi-tive selection cassette for the selection of integrationinto the ES cell genome and a negative selectioncassette for selection of Cre-mediated recombinationof a transgene into the ES cell genome. In one study awild-type loxP site and a mutant loxP511 site, whichdo not efficiently recombine with each other, wereintroduced into the coding region of the liver-spe-cific gene, Mhl-1 , flanking a neomycin or hygromy-cin positive selection gene and the thymidine kinasenegative selection cassette (Figure 2a) (Soukharevet al. 1999). Reporter transgenes, flanked by similar loxP sites, were introduced into these modified EScells with a Cre recombinase expression vector.Following two Cre-mediated cross over events, thetransgenes recombined into Mhl-1 and were identi-fied by selecting for the loss of the thymidine kinasegene with gancyclovir. In this study, it was the au-thors intent to utilize the promoter of Mhl-1 to drive

tissue-specific expression; however, transgeneslinked to exogenous promoters could also berecombined into genomic loxP sites (Feng et al.1999).

Unfortunately, selection schemes that rely onnegative selection cassettes can produce false posi-tives and make the characterization of transgenic EScell clones an inefficient process (Feng et al. 1999;Soukharev et al. 1999). Therefore, a second methodof selecting for Cre/ loxP- mediated recombination oftransgenes into the ES cell genome has been devel-oped. In this instance a cassette containing a loxP

site, a functional selection gene and a neo genelacking a promoter and translational start codon ( neo3half) is introduced into the ES cell genome byrandom integration or homologous recombination(Call et al. 2000; Fukushige and Sauer 1992; Kybaet al. 2002; Le et al. 2003; Wutz et al. 2002). Thetransgene is constructed to contain a promoter andstart codon for neo ( neo 5half) proximal to a loxPsite. Only the precise Cre/ loxP -mediated recombi-nation of the transgene into the ES cell genome willproduce a functional neomycin resistance gene, thusproviding a highly efficient selection system.

Cre/ loxP -recombination has been utilized tointroduce circularized YACs into the ES cell genome(Figure 2b) (Call et al. 2000; Cocchia et al. 2000).YACs were circularized by homologous recombina-tion in the yeast host and modified to contain a neo5half site (Call et al. 2000). These modifications

allowed for the integration of the YAC sequence intothe ES cell genome by a single crossover event andselection of Cre-mediated integration with G418.Interestingly, as with random integration of YACsand BACs, circularized YACs recombined into theES cell genome by Cre-mediated recombination weresubject to deletions and rearrangements prior to orduring integration (Call et al. 2000).

While BAC and PAC vector backbones contain loxP sites and are particularly suited for Cre/ loxP -mediated integration into the ES cell genome, mostdo not contain a means of selection in mammaliancells. Therefore, the use of most BAC/PAC cloneswould be limited to RMCE schemes with a thymi-dine kinase negative selection cassette at the targetlocus unless modified prior to Cre/ loxP -mediatedrecombination. However, at least one PAC vectorcontains mammalian selection genes that couldprovide selection for the Cre/ loxP -mediated inte-gration of a BAC into the ES cell genome (Frengenet al. 2000). This PAC has been constructed to con-tain a blasticidin resistance gene for selection afterintegration into the ES cell genome and a neo 5halfsite in the vector backbone that would function withthe neo 3half sites employed with some of the Cre/

loxP -mediated recombination schemes (Frengenet al. 2000).The Cre/ loxP system provides an effective

mechanism by which single copy transgenes can beintroduced into the same locus of the mouse genomeby a precise recombination event. Furthermore, withhomologous recombination of the target loxP site(s)into an appropriate surrogate site within the ES cellgenome, differences due to position effects can beavoided. To this end, various methods have beendeveloped to target loxP sites to the Hprt locus (Kybaet al. 2002; Le et al. 2003; Wutz et al. 2002). Because



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the Hprt locus is ubiquitously expressed in all tis-sues and, therefore, should have minimal influence

on transgene expression driven by an exogenouspromoter, it is an ideal surrogate site for Cre/loxP-mediated integration of transgenes into the ES cellgenome (Kim et al. 1986; Patel et al. 1986).

There are several disadvantages inherent to theCre/ loxP system that must be considered. First andforemost, the efficiency of inserting a large transgeneinto the ES cell genome by Cre-mediated recombi-nation is extremely low and requires a selectionscheme, such as neo half sites, to efficiently identifyproper recombination events (Call et al. 2000; Coc-chia et al. 2000). Second, the introduction of loxP

sites into the genome by homologous recombinationor random integration requires the selection and

establishment of parental ES cell line subclones. It ispossible that undesired genomic changes couldaccumulate during the subcloning process (Maginet al. 1992). While proper care of ES cell lines inculture minimizes these effects, it is still necessaryto screen loxP -modified subclones for their ability tocontribute to the germline of chimeric mice. Fur-thermore, when random integration is utilized tointroduce loxP sites into the ES cell genome, it isnecessary to carefully screen these lines for the siteof integration and the possible influence of that siteon transgene expression. Although the effect of the

Coding RegionPromoter

hygPromoter tk

lacZ

lox P

lox P

lox P511

lox P511

ATG

lacZ Promoter

ATG

Homologous targeting;hygromycin selection

Recombinase-mediated cassetteexchange; gancyclovir selection

Endogenouslocus

Modifiedlocus

Cre-mediatedknock-in

hyg 3 Neo

hyghyg

lox replacement;G418 selection

CircularizedYAC

Modi

ATG


hygPromoter tk

lacZ

lox P

lox P

lox P511

lox P511

ATG

lacZ Promoter

ATG

Homologous targeting;hygromycin selection

Endogenouslocus

Modifiedlocus

hyg 3 Neo

hyghyg

ATG


hygPromoter tk

lacZ lox P lox P511

lacZ Promoter

ATG

hyg 3 Neo

hyghyg

ATG

5 Neolox P

hyg 3 Neo

hyg

Neo R expression

hyg

fied locus

Cre-mediatedknock-in

ATG

a

b

Fig. 2. Cre /lox P-mediated recombination ofexogenous sequences into the ES cellgenome . (a) Recombinase-mediatedcassette exchange to knock-in a lacZ

reporter construct into the ES cell genome.Using an exogenous promoter, the lox Ptarget site can be introduced into the EScell genome by homologous recombinationor random integration. As depicted, the lox P sites are introduced into the genomeby homologous recombination andexpression of lacZ is dependent on anendogenous promoter. Selection of the Cre-mediated replacement event is dependenton the loss of thymidine kinase ( tk )expression and sensitivity to gancyclovir.(b) A single lox P recombination event tointegrate a circular YAC into the ES cellgenome. The lox P target site in the genome

can be introduced by homologousrecombination or random integration. Thegenomic loxP site is juxtaposed to a neo3half site and the circular YAC has beenmodified to contain a neo 5half site forselection of the appropriate Cre-mediatedrecombination event in G418. Thegenomic insert is represented as a dashedline. Figures is not to scale and adaptedfrom Soukharev et al. 1999 and Call et al.2000.



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locus on different transgenes should be equivalent, itis possible that the locus in which the loxP site isintegrated so severely inhibits or alters transgeneexpression that the transgenes are no longer func-tionally relevant. In addition, with the possibility ofinsertional mutagenesis occurring upon randomintegration of the target site into the genome, it isalso necessary to screen mice derived from the EScell subclones for phenotypes resulting from theintegration event itself.

Homologous recombination into the ES cell genome. In the mid 1980s, it was first demonstratedthat cultured mammalian somatic cells were com-petent to mediate homologous recombination be-tween two independent copies of DNA (Doetschmanet al. 1987; Kucherlapati et al. 1984; Lin et al. 1985;Rubnitz and Subramani 1986; Smithies et al. 1985;Song et al. 1987; Thomas et al. 1986). The discoverythat murine ES cells possess the enzymaticmachinery necessary to mediate homologous

recombination opened the door to developing micecontaining precise genetic alterations (knock out andknock in mice) (Doetschman et al. 1987). Whilehomologous recombination in the ES cell genomeinitially focused on these genomic alterations, it alsoprovided a system for the generation of single copy,chosen-site transgenic mice. Homologous recombi-nation of a single copy of a transgene into the mousegenome eliminates the variables of copy number andinsertional mutagenesis observed with randomintegration. Additionally, with the selection of anappropriate surrogate locus that exerts minimal

influence on transgenic promoters, transgeneexpression in the absence of position effects can beachieved.

Hprt is ubiquitously expressed and is positionedwithin a region of open chromatin (Caskey and Kruh1979; Kim et al. 1986; Patel et al. 1986); therefore, thelocus should exert minimal influence on transgenicpromoters and should not induce heterochromatini-zation of the introduced sequences. The Hprt locushas been used as a surrogate site for the homologousrecombination of small recombinant transgenes intothe ES cell genome for the production of transgenicmice (Table 1). In this method, a transgene is flankedby regions of homology to the partially deleted Hprtlocus of ES cell lines with the E14Tg2a deletion, aswell as the sequences necessary to complement theHprt deletion (Figure 3) (Doetschman et al. 1987;Hooper et al. 1987; Kuehn et al. 1987; Magin et al.1992; Misra et al. 2001; Tsuda et al. 1997; Wu andMelton 1993). Homologous recombination insertsthe transgene into the Hprt locus and reconstitutes

Hprt . Only ES cells that have a transgene properlytargeted to the Hprt locus are able to survive directforward selection with hypoxanthine-aminopterin-thymidine (HAT) medium, for Hprt activity.

Important for position effects, the transgenicsequences are placed upstream of the Hprt promoter,avoiding any potential interactions between the Hprtpromoter and the promoter/enhancer system of thetransgene. Housekeeping promoters that giveunpredictable expression when integrated randomlyconfer broad transgene expression at the Hprt locus,while tissue-specific promoters give the expected

Table 1. Transgenic constructs targeted to the Hprt locus of ES cells

Construct* Tissue Expression Reference

Actin-Bcl-2 Ubiquitous Bronson et al. 1996;Snow et al. 2003

Ferrochelatase-EGFP Erythrocytes Magness et al. 2000Angiotensin (genomic) Liver & Kidney Cvetkovic et al. 2000

Tie2-LacZ Endothelial development Evans et al. 2000eNOS-LacZ Cardiomyocytes and vascular smooth muscle Guillot et al. 2000a -MHC-LacZ Cardiomyocytes Misra et al. 2001vWF-LacZ Heart, skeletal muscle and brain Minami et al. 2002Flt-1-LacZ All vascular beds except liver Minami et al. 2002Angiotensin mutations (genomic) Liver, kidney and diaphram Cvetkovic et al. 2002Tie2 (mutated)-LacZ Loss of expression in vascular beds Minami et al. 2003MBP-LacZ Glial cells Farhadi et al. 2003a -MHC-Gal4 Cardiomyocytes Habets et al. 2003cis -NF- j B-EGFP Fibroblasts, hepatic stellate cells, splenocytes Magness et al. 2004a -MHC-sarcolipin(FLAG) Cardiomyocytes Asahi et al. 2004SRF-LacZ Proepicardial organ Nelson et al. 2004cGT-AT 1a Epithelial cells of the proximal tubule Le et al. 2004U6-shA1 (RNAi bfl-1 / A1 ) Inducible-ubiquitous Oberdoerffer et al. 2005

*The promoters and expressed genes are shown. MHC = myosin heavy chain alpha, vWF = von Willebrand factor, MBP = myelin basicprotein, SRF = serum response factor, cGT = c-glutamyl transpeptidase, AT 1a = angiotensin receptor type 1a, U6 = polymerase III.



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restricted expression when inserted at the Hprt locus(Table 1). However, this may depend on the nature of

the cis -sequences used. In particular, expression ofdevelopmentally regulated genes dependent onchanges in chromatin organization are unlikely to beaccurately expressed by recombinant promoter/cDNA combinations even when integrated at theHprt locus.

BAC transgenes have also been targeted to theHprt locus of ES cells (Heaney et al. 2004). In this

method, a BAC is first modified by in vitro Cre/ loxPrecombination to contain the sequences necessaryfor recombination into the Hprt locus (Figure 4a).Following verification of proper Cre/ loxP -mediatedmodification, and BAC DNA purification and line-arization with a rare-cutting yeast homing endonu-

3 4 5 6 7 8 9

HAT Selection

Transgene

Transgene

h1

2

3 4 5 6 7 8 9

hP

hP 2

h1

35 arm

5 arm

36 kb deletion

( )3 4 5 6 7 8 9

HAT Selection

Deleted Hprt locus

Targeting construct

Transgene

Transgene

Targeted and functional Hprt locush1

2

3 4 5 6 7 8 9

hP

hP 2

h1

35 arm

5 arm

36 kb deletion

( )Fig. 3. Strategy for generating a targetedtransgene at the Hprt locus of ES cell lineswith the E14Tg2a deletion. Recombinationof a transgene into the Hprt locus by areplacement ( W-type) event mediated by

two crossovers. Recombination restoresHprt expression and function and inserts asingle-copy of a transgene into the ES cellgenome. The E14TG2a mutation at theHprt locus (top), a transgene targetingconstruct (middle) and the corrected Hprtlocus containing the single copy transgene(bottom) are shown. The human promoter(hP), human exon 1 (h1), and murine exons(shaded boxes) of the Hprt sequence areshown. Figure is not to scale.

BAC-modifying fragment

2hP h1 3

Amp R

I-Sce I

loxP loxP

Cm R

genomic insert

BAC clone

loxP

5 Arm

hP

genomic insert

Cm RModified BAC

clone

Amp R

I-Sce I

3

h12

cre/ loxP -mediatedrecombination

a

5 Arm

loxP Amp R

2hP h1 3

loxP

5 Homology

Cm R

genomic insertBAC vector

2hP h1 3

Amp R

I-Sce I

loxP loxP

Cm R

genomic insert

BAC clone

loxP

5 Arm

hP

genomic insert

Cm RModified BAC

clone

Amp R

I-Sce I

3

h12

cre/ loxP -mediatedrecombination

b

5 Arm

loxP Amp R

2hP h1 3

loxP

5 Homology

Cm R

genomic insertBAC vector

Fig. 4. Modification of a BAC for homologous integration into the Hprt locus of ES cells. ( a) In vitro Cre-mediatedrecombination of the pJDH6b BAC-modifying fragment into a BAC. Two sequential recombination events integrate themodifying fragment into the BAC. ( b) Linearization of the BAC with I-Sce I results in the flanking of the BAC sequencewith the Hprt homologies in an orientation that favors a replacement recombination event at the Hprt locus. The humanpromoter (hP), human exon 1(h1), and mouse exons are indicated. Figure is not to scale.



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clease, the BAC is introduced into ES cells by elec-troporation (Figure 4b). As expected, BAC transgenesrecombine into the Hprt locus as a single copy andare expressed at the appropriate developmentalstages and in the appropriate tissues both in vitroand in vivo . Although the increased size of BACtransgenes does decrease the efficiency of thehomologous recombination event (approximately a10-fold decrease in the frequency of HAT-resistantcolonies as compared to the Hprt targeting vector),the direct forward selection in HAT medium allowsfor the identification of the rare recombinationevents. Additionally, as observed with both randomintegration and Cre-mediated recombination of largetransgenes into the mouse genome, homologousrecombination at the Hprt locus results in approxi-mately 50 % of HAT resistant ES cell clones con-taining an intact BAC transgene properly targeted to

the Hprt locus, with the remainder containingdeletions or rearrangements of the starting BACsequence.

Targeting large transgenes to the Hprt locus hasseveral advantages over other alternatives to intro-ducing large transgenes into the mouse genome.While Cre-mediated loxP recombination requiresthe establishment of ES cell lines containing a loxPsite and the verification of germline competence,targeting transgenes to the Hprt locus requires onlyone genetic modification event in one of severalestablished germline competent cell lines with theE14TG2a deletion. In addition, unlike transchro-mosomes, BAC transgenes integrated into the Hprtlocus are stably maintained in both the ES cell andmouse genome, as is observed with other largeconstructs randomly integrated into the mousegenome and small recombinant transgenes targetedto Hprt (Bronson et al. 1996; Heaney et al. 2004;Kaufman et al. 1999; Misra et al. 2001; Yang et al.1997).

As with the other alternatives to random inte-gration, there are drawbacks to targeting a BACtransgene to the Hprt locus that must be considered.First, Hprt is X-linked, and known to undergo

random X-inactivation. There is indirect evidence tosuggest that transgenes integrated upstream of thisgene by homologous recombination also undergoinactivation. For some applications, this couldcomplicate the interpretation of transgene functionin female mice as homozygous female mice wouldneed to be generated to assure obligate transgeneexpression in all cells. However, in some instances,mosaic expression can improve the survival of ani-mals with detrimental transgenes or reveal inter-esting aspects of the transgene function.Additionally, due to the significant heterochromat-

inization of sex chromosomes during spermatogen-esis, the effects of X-linked transgenes cannot bedetermined.

Second, it remains unclear if there is an uppersize limit for the efficient homologous recombina-tion of heterologous sequence flanked by smallarms of homology into the mouse genome. Severalrecent studies have relied on the recombination ofgenetically altered BACs (approximately 60 100kb) into their respective genomic loci to generateknockout mice (Testa et al. 2003; Valenzuela et al.2003; Yang and Seed 2003). Unfortunately, thisdoes not necessarily model the insertion of a largestretch of heterologous sequence into a genomiclocus by homologous recombination. There is littleevidence to suggest the length of the interveningsequence between homology arms significantly al-ters recombination frequency (Galli-Taliadoros et

al. 1995; Li and Baker 2000). However, due to thesize limitations of conventional cloning strategies,the ability to study the influence of interveningheterologous sequences on recombination has beenlimited. While the length of the BAC transgenesdoes result in an overall reduction in recombina-tion efficiency, BACs up to 145 kb in size havebeen recombined into the Hprt locus with no ob-servable decrease in targeting efficiency as com-pared to smaller BAC transgenes (Heaney et al.2004).

Conclusion As an increasing number of laboratories around theworld embrace the mouse as a model of humandevelopment and disease, the need for more sophis-ticated approaches to genome modification in-creases. Artificial chromosome-based transgenesiscan be used to transfer entire genomic loci in a singlecontinuous piece, to identify genes responsible forrecessive phenotypes by complementation or domi-nant phenotypes by acquisition, to generate reportersfor tracking gene expression, and to implementsubtle changes in regulatory or structural sequences

in a near endogenous context.

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Artificial Chromosome Based Trans Genes in Genome Function Studies

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