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Mouse genomic technologies: Engineering chromosomal rearrangements in mice
Author: Yuejin Yu
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"780 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS Strategies have recently been developed to intro- duce defined chromosomal rearrangements into the mouse genome by engineering them in embryonic stem (ES) cells using the Cre/loxP site-specific recom- bination system 12 (BOX 1). Using these strategies, mouse models that accurately recapitulate human chromosomal rearrangements have been devel- oped 13?18 . These engineered mouse models, together with the availability of the human genome sequence, will significantly enhance our ability to uncover the specific molecular mechanisms of the defects caused by human chromosomal rearrangements. Chromosomal engineering technology has also led to the generation of novel genetic reagents for the functional analysis of the mouse genome. Deletion chromosomes that are visibly marked by, for exam- ple, coat-colour markers, can be engineered to pro- vide SEGMENTAL HAPLOIDY in the diploid mouse genome. Recessive mutations that are induced in these dele- tion intervals from mutagenesis experiments can be detected by crossing mutant mice to mice that are hemizygous for different regions of the genome 12,19 . Mouse BALANCER CHROMOSOMES have also been devel- oped using Cre/loxP technology by tagging chromo- somal inversions with recessive lethal mutations and coat-colour markers 20 . As inversions suppress recom- bination, these balancer chromosomes can be used to The mouse has become an important model for study- ing genetics and disease because it shares physiological, anatomical and genomic similarities with humans. In both organisms, alterations of chromosomal structure can occur spontaneously or after exposure to specific DNA-damaging agents, causing, in many cases, signifi- cant biological consequences. In humans, chromosomal abnormalities are a principal cause of fetal loss and developmental disorders 1,2 , and chromosomal translo- cations are involved in the genesis of many types of human tumour 3 . Chromosomal rearrangements in mice can be used to model these diseases and enable the fine genetic dissection of their causes. Chromosomal deletions 4,5 , duplications 6 ,inver- sions 7 and translocations 8 can be induced in mice by using radiation or chemical mutagens, such as chlo- rambucil 5 . Some useful rearrangements have been induced using these approaches, one of which has served as a mouse model of trisomy 21 (REFS 6,9). Deletions that overlap a handful of mouse chromoso- mal loci, such as the albino and pink-eyed dilution loci on chromosome 7, have been used for fine genetic mapping and genetic screens 10,11 . However, the useful- ness of radiation or chemical mutagens for inducing rearrangements is limited by the fact that the end points of the induced rearrangements cannot be predetermined. ENGINEERING CHROMOSOMAL REARRANGEMENTS IN MICE Yuejin Yu* and Allan Bradley ? The combination of gene-targeting techniques in mouse embryonic stem cells and the Cre/loxP site-specific recombination system has resulted in the emergence of chromosomal-engineering technology in mice. This advance has opened up new opportunities for modelling human diseases that are associated with chromosomal rearrangements. It has also led to the generation of visibly marked deletions and balancer chromosomes in mice, which provide essential reagents for maximizing the efficiency of large-scale mutagenesis efforts and which will accelerate the functional annotation of mammalian genomes, including the human genome. *Program in Developmental Biology, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. ? The Sanger Centre, Hinxton, Cambridge CB10 1SA, UK. e-mails: yyu@bcm.tmc.edu; abradley@sanger.ac.uk SEGMENTAL HAPLOIDY When a diploid organism is haploid for a certain chromosomal region after its deletion or loss. BALANCER CHROMOSOME A chromosome with one or more inverted segments that suppress recombination. They are used as genetic tools because they allow lethal mutations to be maintained without selection. MOUSE GENOMIC TECHNOLOGIES � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | OCTOBER 2001 | 781 REVIEWS are designed to amplify these markers can be used to isolate genomic clones for constructing the end-point targeting vectors 22 . Genes might also be used as end points using high-resolution mapping information that is available for the mouse genome 23 (see link to Genetic and physical maps of the mouse genome at the Whitehead Institute). Both SSLP markers and genes have successfully been used as the end points for engi- neering numerous chromosomal rearrangements 12,24,25 . In the first step of Cre/loxP-mediated chromosomal engineering, a loxP site, a POSITIVE SELECTION cassette, one of two complementary but non-functional fragments of a hypoxanthine phosphoribosyl transferase (Hprt)gene 12 are introduced into the first end point, in the ES-cell genome, by gene targeting (FIG. 2). To accomplish this, gene-targeting vectors, such as those shown in FIG. 2 are required. These targeting vectors can either be generated in the conventional way, by sequentially inserting various genetic components into a plasmid construct 12,26 , or they can be isolated directly from genomic libraries of pre- made targeting vectors 19 . The targeting vectors from these libraries contain all the genetic elements that are required for chromosome engineering, as illustrated in FIG. 2, they require a minimal amount of manipulation before use and they are available from A.B. ES-cell clones with a loxP site targeted to a first end point can be identified by positive selection and by Southern blot analysis 27 . The subsequent procedures used in our laboratory for generating deletions, duplica- tions and inversions are outlined in FIGS 3 and 4. Only ES-cell lines with an inactivated Hprt gene, such as the AB2.2 line 28 , can be used in these procedures (see link to the Cell Line Request Form for more information on accessing these ES-cell lines). This is because the Cre/loxP-mediated recombination event generates a functional HPRT MINIGENE, which is used to select ES-cell clones that contain the desired rearrangement (see below). After isolating the clones targeted at a first end point, a second loxP site and the complementary Hprt fragment are targeted to a second end point. About six to eight double-targeted clones need to be identified by using the positive selectable markers in the second tar- geting vector and by Southern blot analysis. We expect half of these clones to be targeted on the same chromo- some (in cis) as the original targeted insertion, whereas the other half will be targeted to the homologous chro- mosome (in trans). The type of chromosome rearrangement derived from double-targeted cells will be determined by the loxP configuration (see supplementary Table 1 online for the possible outcomes of Cre-mediated recombina- tion), which depends on the orientation of the loxP site in a targeting vector. To induce loxP recombination, a cre-expression vector, such as pOG231 (REF. 29), is elec- troporated into double-targeted clones. Recombination between the loxP sites unites the 5?Hprt and 3?Hprt cas- settes and reconstitutes a functional Hprt gene. Culturing these ES cells in a medium that contains hypoxanthine, aminopterin and thymidine (HAT) selects for clones that carry the functional Hprt gene, and therefore the rearranged chromosomes. prevent CROSSING OVER in an inverted region ? a prop- erty that can be used to facilitate large-scale mutage- nesis screens 21 (as discussed in more detail below). In this review, we discuss the experimental strategies associated with these recent advances and the contri- butions that mice with engineered chromosomes are making to functional genomics, and to the study of human genetics and diseases such as cancer. Chromosomal engineering The strategy of chromosome engineering is based on the techniques of gene targeting in ES cells and the Cre/loxP system (FIG. 1). Using gene targeting, two loxP sites are inserted sequentially into two loci in the ES- cell genome. The transient expression of the gene that encodes Cre recombinase in double-targeted ES cells induces recombination between the two targeted loxP sites to generate the rearranged chromosome. Using various methods, such as drug selection, Southern blot analysis and fluorescent in situ hybridization (FISH), the ES-cell clones that carry the desired chromosomal rearrangement are identified and characterized. Chimaeras are generated by injecting these ES cells into mouse BLASTOCYSTS, from which the progeny that carry engineered chromosome are derived (FIG. 1). Deletions, duplications and inversions. Defined chro- mosomal deletions, duplications and inversions are important rearrangements not only because they con- stitute prevalent classes of genomic anomaly in humans, but also because they provide powerful reagents for mouse functional genomics. Generating these types of genomic alteration begins with defining the two end points of the rearrangement. For regions greater than 1 Mb, end points might be selected from more than 6,000 simple sequence length polymor- phism (SSLP) markers that have been mapped in the mouse genome (see link to STS Physical Map of the Mouse at the Whitehead Institute). The primers that CROSSING OVER The exchange of genetic material between two homologous chromosomes. BLASTOCYST A preimplantation embryo that contains a fluid-filled cavity called a blastocoel. POSITIVE SELECTION When a specific chemical is added to a culture medium, the cells that express a positive selectable marker gene, such as the neomycin or puromycin resistance genes, survive and are selected for. HPRT MINIGENE (Hypoxanthine phosphoribosyl transferase gene). This is divided into two complementary, but non- functional, fragments: 5?Hprt contains exons 1?2 and 3?Hprt contains the remaining exons, 3?9. Each Hprt fragment is linked to a loxP site, and Cre- mediated recombination unites the 5? and 3? cassettes, and restores Hprt activity, which is required for purine biosynthesis and allows desired recombination events to be selected for in HAT (hypoxanthine, aminopterin and thymidine) medium. Box 1 | Cre/loxP site-specific recombination The reaction catalysed by the P1 bacteriophage Cre recombinase leads to site- specific recombination between two loxP sites 51,52 . The loxP sequence consists of two 13-bp inverted repeats and an 8-bp asymmetrical core spacer region, which determines the orientation of the site (as shown). The recombination reaction is initiated by Cre binding specifically to the inverted repeat sequences at loxP sites, which leads to the formation of a synapse that consists of four Cre subunits and two loxP sites in the same orientation. Cre catalyses exchange between the pair of sites in the core spacer region by concerted cleavage and rejoining reactions. A cis recombination event between two loxP sites in the same orientation will lead to the excision of the loxP-flanked DNA sequence as a circular molecule. If loxP sites are orientated in opposite directions, the loxP-flanking sequence will be inverted. Recombination between two loxP sites in trans will lead to the reciprocal exchange of the regions that flank the loxP sites. Cre can also induce these recombination events when the loxP sites are located several megabases apart on the same chromosome, or on two homologous or non-homologous chromosomes 12,24,38,39 . Inverted repeat 5' ? ATAACTTCGTATAGCATACATTATACGAAGTTAT ? 3' 3' ? TATTGAAGCATATCGTATGTAATATGCTTCAATA ? 5' Core region Inverted repeat � 2001 Macmillan Magazines Ltd 782 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS and orders of the selection cassettes are unknown is considerable (see FIGS 3 and 4, and supplementary Table 1 online). To obtain clones that carry a chromosomal dele- tion and/or a chromosomal duplication, the two tar- geted loxP sites should be orientated in the same direction. If the loxP sites are orientated in opposite directions, Cre-mediated recombination between loxP sites in cis and in trans will generate an inversion and inviable recombination products (ACENTRIC and DICENTRIC chromosomes), respectively (FIG. 4 and sup- plementary Table 1 online). Therefore, if after cre expression, HAT-resistant colonies are not recovered from some of the double-targeted clones, this usually indicates that the two targeted loxP sites are located in opposite orientations. Given such an observation, deletions and duplications can be generated by inverting the loxP selection cassette and re-targeting the second end-point vector. Cre-mediated recombination can occur in a cell at the G1 phase of the cell cycle or after DNA replication has occurred (S/G2). After chromosome replication, four loxP sites will be present in the double-targeted ES- cell genome, and Cre-mediated recombination can occur between sister or non-sister chromatids depend- ing on whether the loxP sites are inserted in cis or in trans. These post-replication events might lead to sever- al recombination outcomes (as shown in FIGS 3 and 4, and supplementary Table 1 online), some of which will not survive selection in HAT medium. We have found that, when the cre expression vector ? pOG231 ? is used, the efficiency of Cre-mediated cis recombination is ~10% and does not alter appre- ciably if the distance between two targeted loxP sites is changed from a few kilobases to up to 10 Mb (REF. 25) (Y.Y. and A.B., unpublished data). When the loxP sites are on homologous chromosomes (trans), recombi- nation is approximately two to three orders of magni- tude less efficient 25 than when they are in cis.So,when loxP sites are believed (or known) to be in the same orientation, it is possible to identify double-targeted clones in which the loxP sites are inserted in cis or in trans by analysing recombination efficiencies. Selection analysis of the HAT-resistant clones with G418 and puromycin can also be used to classify the clones that carry various types of chromosomal rearrangement. These rearrangements can then be further analysed by Southern blot analysis and by FISH using mouse bacterial artificial chromosome (BAC) clones as probes. The same strategy can be used to generate inver- sions (FIG. 4), although in this case the two loxP sites remain at the end points of the rearrangement after an inversion has been generated. In principle, the inverted region could revert back to its non-inverted state; however, although this might occur in a small percentage of cells, these cells will not survive in the HAT selection medium. Variations of the aforementioned strategy have been reported by other groups 24,30?32 . Besides pOG231, several other cre-expression vectors, such as pBS185 If the relative orientations of the two end-point loci (with respect to the centromere) are known, a specific loxP configuration can be designed. If the orientations of the two loci are unknown, as is the case for many chromosome-engineering projects, targeting vectors with different orientations of loxP sites will need to be tested (FIGS 3 and 4). The complexity of the recombina- tion products that are generated when the orientations ACENTRIC A chromosome or chromatid without a centromere. DICENTRIC A chromatid or a chromosome that has two centromeres. K14?AGOUTI A transgene in which the agouti gene is under the control of the keratin 14 promoter. Its expression produces a yellowish coat colour in mice. a b c d e f g Generate progeny that carry the engineered chromosome Identify chimeric mice Inject ES cells into blastocyst Identify the ES-cell clones with the desired rearrangements Isolate double-targeted clones Insert loxP site into first end point Insert loxP site into second end point on same chromosome or on different chromosome Introduce cre Figure 1 | A general strategy for chromosomal engineering in mice. a | A loxP site is inserted into the first end point in the embryonic stem (ES)-cell genome using a targeting vector that carries a positive selectable marker gene. b | A second loxP site, linked to a different positive selectable marker gene, is targeted to the second end point, either on the same chromosome or on a different chromosome by gene targeting or by random insertion. c | The expression of cre in double-targeted ES cells catalyses recombination between loxP sites at the rearrangement end points. d | ES-cell clones that carry the desired chromosomal rearrangements are identified and molecularly characterized. e | The selected ES cells are injected into mouse blastocysts and the embryos are transferred into the uteri of pseudopregnant foster mothers. f | Chimaeras that are generated from blastocyst injection are mated with wild-type mice to establish germ- line transmission of the modified genome. g | The progeny derived from the chimaeras are characterized, and a mutant mouse line that carries an engineered chromosome is established. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | OCTOBER 2001 | 783 REVIEWS Nested chromosomal deletions. These are a series of overlapping deletions that surround a predetermined genomic locus. These deletions vary in size and have different end points (nested end points), but many of them will overlap. If the genomic locations of the end points are known, nested deletions can be extremely useful for mapping novel recessive mutations. By crossing mice that carry a hemizygous deletion with a mouse line that carries a novel recessive mutation, progeny that harbour both the deletion and the novel mutation in trans can be generated. If a recessive mutant phenotype is observed in the progeny of such a cross, it indicates that the chromosome that carries the deletion cannot complement the novel mutation; the novel mutation is therefore located in the deletion interval. Using this approach, novel mutations can be rapidly mapped to a specific deletion interval by cross- ing mutant mice with mice that carry nested deletions. To efficiently engineer these types of reagent, we have developed an approach for constructing deletion com- plexes that does not require that targeting vectors be made for the nested end points 33 . Deletion complexes can be anchored to a predetermined location in the genome by targeting the 5?Hprt?loxP cassette as described previ- ously. The 3?Hprt?loxP cassette is then inserted randomly into the ES-cell genome by retrovirus-mediated integra- tion (FIG. 5), which generates a library of ES clones with the same targeted end point and a collection of random end points. Only a subset of random insertions will occur on the same chromosome as the original targeting event. However, Cre/loxP recombination efficiency is several orders of magnitude more efficient when loxP sites are inserted on the same chromosome. So, after the expres- sion of cre, most HAT-resistant clones will be derived from retroviral insertions that have occurred in cis to the targeted insertion. Cre/loxP recombination efficiencies will also decrease if the sites are separated by more than 10 Mb. So, most HAT-resistant clones will have rearrangements that are less than 10 Mb. Clones that are generated by using this strategy carry a random distribu- tion of deletion sizes that range from a few kilobases to several megabases 33 . Clones that contain chromosomal deletions lose the neo and puro cassettes, and so can be distinguished from other types of rearrangement by sib-selection in G418 and puromycin. This nested deletion strategy has also been repeated using electroporation to insert the loxP cassette ran- domly into the ES-cell genome 34 . Compared with retro- virus-mediated integration, insertion by electroporation might increase the risk of genomic rearrangements occuring at the insertion site and tandem repeats of a vector might be introduced into the insertion site, although these should be reduced to a single locus by the activity of Cre on a head-to-tail concatenate. Deletions that are generated by the random inser- tion of the second end point are usually characterized by Southern blot analysis and by FISH and, if a HYBRID ES-CELL LINE is used, by SSLP analysis. The end points can be defined by cloning the genomic DNA that flanks the deletion end points and by mapping these junction fragments onto a physical map of the region. (REF. 24), pBS500 (REF. 30) and pIC-CRE 32 ,have been used in chromosomal-engineering experiments in ES cells. Other strategies also select for the desired recom- bination products in different ways. In some cases, a herpes simplex virus thymidine kinase (HSVTK) gene, which acts as a NEGATIVE SELECTABLE MARKER, is inserted between the rearrangement end points. Cre/loxP- mediated recombination events that result in a dele- tion and the loss of this marker can be selected by cul- turing ES cells in medium that contains 1,2? deoxy-2?-fluoro-?-D-arabinofuranosyl-5-iodo- uracil 24,30?32 , which kills cells that express HSVtk. However, negative selection cannot be used to isolate duplications or inversions. Positive-selection strategies facilitate the isolation of cells with reciprocal recombi- nation products (such cells would not survive negative selection), which allows ES cells with balanced genetic changes to be recovered. These rearrangements can then be assessed independently of each other after their segregation in the germ line of mice. This is par- ticularly useful because duplications can rescue mice that inherit a corresponding HAPLOINSUFFICIENT deletion. One factor that limits the generation of deletions in ES cells is the size of the rearranged interval. Available evidence indicates that large deletions, such as those deletions larger than 22 cM, might lead to ES- cell lethality or to a severe growth disadvantage of the cells in culture 25 . Although Cre/loxP recombination will occur readily over these large distances, clones will often emerge from these experiments that have undergone a compensatory genetic change, such as a chromosomal duplication 25 . a 5' Hprt vector Genomic locus Targeted locus Ty N5' Hprt TyN 5' Hprt AgP 3' Hprt b 3' Hprt vector Genomic locus Targeted locusAg P P Puromycin resistance gene N Neomycin resistance gene Ag K14?Agouti transgene Ty Tyrosinase minigene loxP 3' Hprt Figure 2 | Gene targeting in embryonic stem cells. Insertional targeting vectors, as shown, can be used to insert loxP sites, positive selectable markers, the Hprt gene fragments and coat-colour markers (such as Ty and Ag) to predetermined loci in the embryonic-stem-cell genome. a | Expression of the neomycin resistance and b | puromycin resistance genes allows different targeting events to be selected. The complementary, but non-functional, 5?Hprt and 3?Hprt fragments are derived from a Hprt minigene 12 . The dark blue and light blue bars represent regions of homology between the vector and the genomic locus. The vector is linearized in the region of homology (gap) to stimulate targeted insertion into the locus. X represents recombination between the vector and the genome. Ag, the K14?AGOUTI transgene; Hprt, hypoxanthine phosphoribosyl transferase; Ty, the TYROSINASE minigene. TYROSINASE Tyrosinase is required for melanin biosynthesis, and the expression of its gene leads to pigment production, and is therefore used as a coat-colour marker. HSVTK The herpes simplex virus thymidine kinase (HSVtk) is essential for thymidine nucleotide biosynthesis through a salvage pathway and is often used as a negative selectable marker in gene targeting. NEGATIVE SELECTABLE MARKER A negative selectable marker gene, such as HSVtk,allows cells that express it to be killed when a specific chemical is added to a culture medium, whereas cells that no longer express the marker gene survive. HAPLOINSUFFICIENCY A phenotype that arises in diploid organisms owing to the loss of one functional copy of a gene. HYBRID ES-CELL LINE An embryonic stem (ES) cell line isolated from F 1 hybrid embryos, such as from crosses between the strains C57BL/6-Tyr c1Brd � 129S7 or 129S1 � CAST/Ei. These lines facilitate simple sequence length polymorphism analysis. � 2001 Macmillan Magazines Ltd 784 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS efficiently; however, they require extensive additional characterization to define each deletion interval. Chromosomal translocations. One of the main reasons for engineering defined chromosomal translocations is to develop mouse models for human translocations, which underlie certain forms of cancer by causing the abnormal expression of cellular oncogenes or by creating novel fusion genes 3 . Nested deletions have also been generated by irradia- tion 35?37 . Deletions that are induced by irradiation can be localized and made selectable by targeting a vector that carries a negative selection cassette (such as HSVtk) to a predetermined locus. Before irradiation, the cells can be cultured under positive selection pressure to retain the tar- geted locus. After irradiation, clones that carry the desired deletion can be identified by loss of the negative selection marker. Using this approach, deletions can be produced Isolate 6?8 double-targeted clones trans Cre G1 G2 HAT selection Cre G1 G2 3' P 5' N 3'P 5'N 3' P P 3' 3' P 5'N 3' P P 5' N N 5' 3' P N 3' 5' P N 3' 5' P P P N N N cis 3'P 5' N 3'P 5'N 5' N 5' 5' 3' 3' 3' 5' P N P N 5' 3' P N N 5' 3'3' P5' N 5' 5' PP 3' 3' N Recombination efficiency (%) Final chromosomal rearrangement in ES cells 0.1 ? 0.01 Drug selection HAT G418 Puro 10 Puromycin resistance gene Neomycin resistance gene T Targeted loxP Df Deficiency Dp Duplication R Resistant S Sensitive5' 3' 3' Hprt 5' Hprt Mouse chromosome 3' P Df/+ Df/+ Df/TDp/TDp/+ Df/Dp Df/Dp Df/Dp Df/Dp R S S R S S R R S R S R R R R R R R R R R R R R R R R Figure 3 | Engineering a deletion and/or a duplication in embryonic stem cells. An experimental procedure for engineering chromosomal deletions and duplications. The cassettes can be targeted in two orientations, only orientations that result in deletions or duplications are illustrated. G1 and G2 indicate the different phases of the cell cycle in which recombination occurs. In G2, four loxP sites are located on duplicated chromatids, and recombination events will result in various products. Drug selection will help with identifying the desired rearrangements. For clarity, only the drug-resistance characteristics of the HAT-resistant clones are shown. After trans recombination in G2, the chromosome that carries the Hprt resistance marker will either segregate with the reciprocal product (carrying the duplication) to give a Df/Dp cell, or it will segregate with a non- recombined chromatid that carries the targeting vector (T). These cells (Df/T or Dp/T) are therefore resistant to either G418 or puromycin (Puro) but not both. HAT, hypoxanthine, aminopterin and thymidine; Hprt, hypoxanthine phosphoribosyl transferase. � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | OCTOBER 2001 | 785 REVIEWS chromosomes is also lower than that obtained when loxP sites are inserted within a few megabases of each other on homologous chromosomes. To generate a fusion protein from a chromosomal translocation, the targeting vectors need to be specially designed so that after translocation, two genes original- ly located on two non-homologous chromosomes can be linked through their introns, with the loxP site embedded in the junction region of the breakpoint. After RNA splicing, an in-frame fusion mRNA and protein are generated as a result (FIG. 6b). To prevent the generation of acentric and dicentric chromosomes, only pairs of genes with the same transcriptional orien- tations relative to their centromeres can be engineered to generate fusion proteins. Mouse chromosomal translocations with predeter- mined breakpoints have been created using Cre/loxP recombination 15,16,38,39 . Translocations are generated when loxP sites are targeted to non-homologous chro- mosomes. To obtain the desired chromosomal translo- cation, these targeted loxP sites need to be orientated in the same direction relative to their respective cen- tromeres. If two targeted loxP sites are in opposite ori- entations, recombination will result in acentric and dicentric chromosomes (FIG. 6a). The efficiency of Cre/loxP recombination between non-homologous chromosomes is several orders of magnitude lower than that of the recombination between loxP sites on the same chromosome. The frequency of Cre/loxP- mediated recombination between non-homologous Isolate 6?8 double-targeted clones transcis Cre G1 G2 HAT selection Cre G1 G2 3' P 5'N 3'P 5' N 3' P 3' P 3'P 5' N 3' 5' 3' 5' P N P N 3' 5' P N 3' 5' P P P N N N 3' 5' 5' 3'P 5'N 3' 3' 5' P N P N 3' 5' 3' 5' PP 3'N 5' N P 3' P N Recombination efficiency (%) R R R Inv/+ Dicentric and acentric HAT G418 Puro 10 R R R Inv/+ 10 N 5' 3' 5' 3' P3'P 5'N 5' N P N Final chromosomal rearrangement in ES cells Drug selection Puromycin resistance gene Neomycin resistance gene loxP R Resistant Inv Inversion5' 3' 3' Hprt 5' Hprt Mouse chromosome Figure 4 | Engineering an inversion in embryonic stem cells. An experimental procedure for engineering chromosomal inversions. G1 and G2 indicate the different phases of the cell cycle in which recombination occurs. Only orientations of the cassettes that result in inversions are illustrated. Cre-mediated recombination at G1 or G2 will result in various products. HAT, hypoxanthine, aminopterin and thymidine; Hprt, hypoxanthine phosphoribosyl transferase; Puro, puromycin. � 2001 Macmillan Magazines Ltd 786 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS chromosomal translocations are crucial events in the formation of many types of human tumour, such as leukaemia, lymphoma and sarcoma 3 . Because there are many conserved linkage groups between the human and mouse genomes, the chromosomal rearrange- ments that are involved in human disease can be mod- elled in mice. These mouse models can be used to study the molecular events that are associated with these diseases. Mouse models of chromosomal dele- tions can also be used to analyse rearranged chromo- somal regions and can facilitate the identification of the genes that are involved in the clinical features of chromosomal disorders. The other main driving force behind recent advances in mouse chromosomal manipulation has been the need to generate resources to facilitate genetic screens 28 . Chromosomal rearrangements, such as visi- bly marked deletions and balancer chromosomes, have been instrumental in the success of genetic screens in Drosophila. In mice, mutagenesis efforts to generate and map recessive mutations have been hindered by the lack of marked deletions in most regions of the genome and by the unavailability of balancer chromo- somes. The creation of these reagents through chro- mosomal engineering technology will change future strategies for large-scale, recessive genetic screens in mice and will facilitate the functional analysis of the mouse genome. Modelling human disease. Among human chromoso- mal rearrangements, deletions constitute an important class. Deletions are often identified when haploinsuffi- cient gene(s) in the deleted region cause a clinical phe- notype. The positive-selection-based Cre/loxP strategy for engineering defined chromosomal deletions is uniquely suited for identifying and analysing mam- malian haploinsufficient loci. Alternative strategies for generating deletions that use negative selection 24,30?32 and irradiation 35?37 suffer from the disadvantage that they do not generate selectable reciprocal products. However, the positive-selection-based strategy allows duplications to be recovered from trans recombination events (see supplementary Table 1 online). Duplications are important experimental tools because they allow haploinsufficient deletions to be maintained, and allow mice that harbour both a dele- tion and the reciprocal duplication to be recovered because they are genetically balanced. Using chromosomal-engineering techniques, key genetic elements in several human chromosomal dele- tion disorders have recently been identified by engi- neering mouse chromosomal deletions in regions that are homologous with those deleted in certain human deletion disorders, such as DiGeorge syndrome and Prader?Willi syndrome 13,14,17,18 . The power of this approach has been illustrated by modelling the deletion that is involved in DiGeorge syndrome. DiGeorge syn- drome is associated with a hemizygous deletion on human chromosome 22, del(22)(q11.2; q11.2). The DiGeorge region had been recalcitrant to molecular dissection in humans 41 . Despite intensive efforts, To induce a translocation in vivo, mice that carry both targeted end points can be crossed with trans- genic mice that express cre under the control of a reg- ulatory element with the desired tissue and temporal specificity 15,16 . This approach not only has been used to generate better models of human leukaemia-asso- ciated translocations, such as the t(8;21)(q22;q22) (REF. 15) and t(9;11)(p22;q23) (REF. 16) translocations, which cause acute leukaemia, but also circumvents the problem of transmitting translocations through the male germ line, as the presence of chromosomal translocations in male germ cells can cause infertility 40 . Recombination events that give rise to chromosomal translocations can reach recombination efficiencies of 10 ?4 to 10 ?6 in tissues that express Cre 15 . Although these recombination rates are low, they can mimic the rare genetic events that are crucial steps in neoplastic transformation (as discussed below). Applications of chromosomal engineering About 0.6% of all newborn human infants have cyto- genetic imbalances 2 , so chromosomal anomalies are a principal cause of human genetic disease. Somatic Recombinant retrovirus Cre 5' N N PLTR LTR3' P abcd 5' 3' ab + + cdLTR LTR 5' 3' bc dLTR a PLTR 5' 3' + cdLTR a P b LTR N N Figure 5 | Nested chromosomal deletions induced with a retroviral vector. The first deletion end point is fixed by targeting the 5?Hprt cassette and a loxP site to a predetermined locus. The 3?Hprt cassette and the second loxP site are then integrated randomly into the embryonic- stem-cell genome using a recombinant retroviral vector. For clarity, only G1 recombination events from retroviral orientations that result in deletions are illustrated. Cre catalyses recombination between the loxP sites (red arrowheads), and HAT medium is then used to select for the clones that carry the recombinant chromosomes. The nested deletions can be identified from a pool of HAT-resistant clones on the basis of their sensitivity to G418 and puromycin. 3?, 3?Hprt; 5?, 5?Hprt; a?d, genetic markers; HAT, hypoxanthine, aminopterin and thymidine; Hprt, hypoxanthine phosphoribosyl transferase; LTR, retroviral long terminal repeat; N, the neomycin resistance gene; P, the puromycin resistance gene. (Modified with permission from REF. 33.) � 2001 Macmillan Magazines Ltd NATURE REVIEWS | GENETICS VOLUME 2 | OCTOBER 2001 | 787 REVIEWS on mouse chromosomes 4 and 16, were used as the end points for Cre-mediated recombination. In t(9;11)(p22;q23), the translocation generates a fusion gene from the genes MLL (myeloid/lymphoid or mixed-lineage leukaemia) and AF9 (myeloid/lym- phoid or mixed-lineage leukaemia; translocated to, 3). To generate such a fusion gene in mice, the mouse Af9 and Mll genes, located on chromosomes 4 and 9, respectively, were used as the rearrangement end points. Mice with double-targeted end points were crossed with transgenic lines that express cre in various organs, including the brain, and the desired rearrange- ments were produced in their progeny. However, cre expression has not yet been targeted to the haematopoietic cell lineages and, possibly as a result, leukaemia has not been reported in the mice that carry these translocations 15,16 . These translocations illustrate a further advantage of the Cre/loxP chromosomal engineering system. Inducing recombination in vivo can generate chro- mosomal deletions, duplications or transloca- tions 15,16,25,43,44 . This approach can often be essential when the rearrangements cause ES-cell lethality 25 or embryonic death 26 , or when modelling human chro- mosomal rearrangements that occur only in certain somatic cell types 15,16 . Engineered chromosomes for functional analysis. Experimental approaches for the functional charac- terization of the genome of an organism rely on the generation of mutations. For the mutational analysis of diploid organisms, such as the mouse, genetic tools, such as marked deletions and inversions, are important reagents because they facilitate rapid genetic mapping and maintenance of randomly gen- erated mutations, such as those generated during ethylnitrosourea (ENU) mutagenesis screens. The development of chromosomal engineering tech- niques has significantly expanded the repertoire of these powerful genetic tools. In an effort to functionally analyse mouse chromo- some 11, 18 deletions have been engineered on this chromosome using Cre/loxP technology 26,33 (Y.Y. and A.B., unpublished data; see also link to the Chromosome 11 deletion map). This work has gener- ated mouse lines that carry regions of segmental hap- loidy, which can be used to screen ENU-mutagenized mice to identify recessive mutations. Smaller nested deletions can then be used for complementation test- ing, to narrow down the genomic location of a muta- tion as a prelude to cloning. Thereafter, the mutated gene can be identified by genomic complementation with BACs 45,46 and/or by sequencing the entire muta- tion-carrying region. Although many deletions have now been generat- ed on mouse chromosome 11, several of these dele- tions are haploinsufficient 26 (Y.Y. and A.B., unpub- lished data). Mice that carry these deletions either die during embryogenesis or show disease phenotypes. This prevents their use in genetic screens, although it does identify regions of the genome that are worthy including the analysis of the finished genomic sequence of chromosome 22 (REF. 42), the gene(s) responsible for the clinical features of this disorder have not been iden- tified using human molecular genetic approaches. An alternative strategy exploiting chromosomal engineering was used to generate a 1.2-Mb deletion, Df(16)1, in the region of mouse chromosome 16 that corresponds to the minimal DiGeorge region on human chromosome 22 (REF. 13). The hemizygous dele- tion mice, Df(16)1/+, develop cardiovascular defects similar to those observed in DiGeorge syndrome patients. Importantly, in mice that harbour both Df(16)1 and the reciprocal duplication, no heart defects are detected, proving that reduced gene dosage in the deleted region is responsible for the mutant car- diovascular phenotype seen in the Df(16)1/+ mice 13 .To locate the gene(s) involved in this phenotype in Df(16)1, smaller overlapping deletions were generated by using known end points or by using randomly gen- erated nested deletions that were induced by a recom- binant retrovirus 33 . Mice that carry these sub-deletions were then analysed for heart defects. These studies nar- rowed down the candidate interval to a region that contained a few genes, one of which ? Tbx1 ? went on to be identified as the haploinsufficient gene that causes the principal cardiovascular defects in DiGeorge syndrome 18 . The same conclusion was reached inde- pendently by a second group that also used chromoso- mal engineering techniques 17 . Progress has also been made in efforts to model human leukaemia-associated translocations, such as t(8;21)(q22;q22) (REF. 15) and t(9;11)(p22;q23) (REF. 16). In t(8;21), the breakpoints of the translocation are located in the genes AML1 (acute myeloid leukaemia 1; also called RUNX1, runt-related transcription factor 1) and ETO (also called CBFA2T1, core-binding factor, alpha subunit 2; translocated to, 1). To model this translocation, the orthologues of these genes, located ENU (N-ethyl-N-nitrosourea). A potent mutagen that primarily generates single-base-pair mutations in mouse spermatogonia germ cells. Inter-chromosomal translocation a b Intron Gene A Intron Dicentric and acentric chromosomes Cre RNA splicing Fusion mRNA Junction of a translocation breakpoint Cre Exon Exon Gene B AB Figure 6 | Engineering chromosomal translocations. A strategy for engineering a chromosomal translocation and an associated fusion gene. a | Cre-mediated recombination leads to chromosomal translocation or dicentric and acentric chromosomes, depending on the relative orientations of the loxP sites (red arrowheads) on two non-homologous chromosomes. b | An in-frame fusion mRNA and protein can be generated by engineering an appropriate junction at the translocation breakpoint. � 2001 Macmillan Magazines Ltd 788 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS number of recessive mutations that could be detected by using it would be significantly reduced. To over- come this problem, we have generated inversion chromosomes. Using engineered inversions for mutagenesis screens has several advantages. First, unlike a deletion, a 20- to 30-cM inversion can be generated without causing a detrimental effect to mice. So, by using an inversion, a much larger genomic region can be screened. Second, a heterozygous inversion effectively suppresses crossing over in the inverted genomic seg- ment because a single crossover between loci in the rearranged interval leads to inviable acentric and dicentric chromosomes or aneuploidy. Therefore, inversions can be used to maintain the genomic integrity of a mutagenized region. Third, an inversion can be designed to function as a balancer chromosome by tagging it with a recessive lethal mutation, which prevents animals that carry a homozygous inversion from being viable. Finally, a coat-colour marker can be added to an inversion chromosome so that its inheri- tance can be followed without requiring the genotypic analysis of progeny. To facilitate the isolation of ENU-induced reces- sive mutations on mouse chromosome 11 (see link to Chromosome 11 ENU mutagenesis programme), the first mouse balancer chromosome was constructed on chromosome 11 using Cre/loxP-mediated recom- bination 20 (FIG. 7a). This balancer chromosome is based on a 24-cM inversion between the Trp53 gene and the Wnt3 gene. Mice that are homozygous for this inversion die during embryogenesis owing to the disruption of the Wnt3 gene, which is required for embryonic development 47 , at one of the inversion end points. In addition, a coat-colour marker, K14?Agouti, has been inserted into the mutated Wnt3 locus. Such a marked balancer chromosome consti- tutes an ideal reagent for the isolation of novel ENU- induced recessive mutations in a three-generation breeding scheme 21 (FIG. 7b). Conclusions and perspectives ES-cell technology has drastically enhanced our ability to engineer various types of mouse genomic alteration, which now include single-gene knockouts, single-base- nucleotide alterations, conditional mutations (see review by Mark Lewandoski on p743 of this issue for more on this technique) and megabase rearrangements. Novel ES-cell-based technologies for genomic manipulation will undoubtedly continue to emerge. The technologies of chromosome manipulation will become easier to apply as the mouse genome sequencing project progresses (see link to NCBI?s mouse genome sequencing page). This is because the availability of a complete mouse genome sequence will facilitate the selection of end points and the con- struction of targeting vectors for use in engineering- defined chromosomal rearrangements. The mouse genome sequence will also help with identifying the location of random integration sites in nested chromosome deletions. of further analysis. A deletion can be generated that encompasses a smaller interval as a way to avoid hap- loinsufficient gene(s), but this reduces the number of genes located in the interval. A mouse line that car- ries a smaller deletion is therefore not an efficient tool for conducting a genetic screen because the Wnt3 Balancer Trp53/Wnt3 Trp53 Trp53/Wnt3 P P N N Ty 3' 5' Ag Ag Ty Wild type G0 Inv/+ � * /+ a b Puromycin resistance gene Neomycin resistance gene K14?Agouti transgene loxP 5' 3' 3' Hprt 5' Hprt Tyrosinase minigene ENU * G1 Stock carrying marker Select yellow mice Inv/Q � � * /Inv * G2 Yellow Non yellow, marked Yellow, marked Lethal Inv/ ** /Q Inv/Q Inv/Inv * G2 intercross Genotype frequency * * * Q QQ G3 Mutant class Carrier Lethal 25% 50% 25% * * * Figure 7 | A mouse balancer chromosome and its use in ENU mutagenesis screens. a | The mouse balancer chromosome, Inv(11)8, is based on an inversion between Trp53 and Wnt3. Inv(11)8 mice carry an engineered coat-colour marker (agouti) and die when homozygous for the inversion during embryogenesis because of the targeted mutation at the Wnt3 locus. b | A breeding scheme to isolate recessive mutations using a balancer chromosome, such as Inv(11)8. In the first generation (G1), mice hemizygous for Inv(11)8 and an induced mutation are generated by mating ENU-treated males (*/+) to females that carry the balancer chromosome (Inv/+), which is marked by the dominant coat-colour marker, K14?Agouti. The G1 mice (*/Inv) are then crossed with mice that carry the balancer chromosome (Inv) and another visible marker (Q) on the homologous chromosome that is distinguishable from K14?Agouti. The Inv/* mice can be visually identified among the G2 mice, and Inv/Q and Q/* mice are not used further. Sibling matings between the Inv/* mice generate two classes of G3 mice, Inv/* and */*, which can be distinguished by the presence of K14?Agouti on the balancer chromosome. If all G3 animals carry K14?Agouti, the induced mutation causes embryonic lethality. Hprt, hypoxanthine phosphoribosyl transferase. (Modified with permission from REF. 21.) � 2001 Macmillan Magazines Ltd major clinical features of the disorder 9,50 . Because human chromosome 21 orthologues have been locat- ed to regions of conserved linkages on mouse chromo- somes 10, 16 and 17, a better mouse model could be engineered that would carry segmental trisomies of all these genomic regions. Chromosomal engineering could also be used to generate small overlapping dupli- cations in mouse chromosomal regions conserved with the trisomic human chromosome 21 regions to identify the crucial genomic domain(s) and causative gene(s) that are responsible for the clinical characteris- tics of the disorder. Chromosomal-engineering technology has increased our ability to manipulate the mammalian genome, which has special significance in the current genomic era. 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A site-directed chromosomal translocation induced in embryonic stem cells by Cre?loxP recombination. Nature Genet. 9, 376?385 (1995); erratum 12, 110 (1996). 39. Van Deursen, J., Fornerod, M., Van Rees, B. & Grosveld, G. Cre-mediated site-specific translocation between nonhomologous mouse chromosomes. Proc. Natl Acad. Sci. USA 92, 7376?7380 (1995). References 38 and 39 introduce the use of targeted Cre/loxP strategies for generating chromosomal translocations in mouse embryonic stem cells. 40. Lyon, M. F. & Meredith, R. Autosomal translocations causing male sterility and viable aneuploidy in the mouse. Cytogenetics 5, 335?354 (1966). 41. Scambler, P. J. The 22q11 deletion syndromes. Hum. Mol. Genet. 9, 2421?2426 (2000). 42. Dunham, I. et al. The DNA sequence of human chromosome 22. Nature 402, 489?495 (1999); erratum 404, 904 (2000). 43. Herault, Y., Rassoulzadegan, M., Cuzin, F. & Duboule, D. Engineering chromosomes in mice through targeted meiotic recombination (TAMERE). Nature Genet. 20, 381?384 (1998). 44. Matsusaka, T. et al. Dual renin gene targeting by Cre- mediated interchromosomal recombination. Genomics 64, 127?131 (2000). 45. Antoch, M. P. et al. Functional identification of the mouse circadian Clock gene by transgenic BAC rescue. Cell 89, 655?667 (1997). 46. Hejna, J. A. et al. Functional complementation by electroporation of human BACs into mammalian fibroblast cells. Nucleic Acids Res. 26, 1124?1125 (1998). NATURE REVIEWS | GENETICS VOLUME 2 | OCTOBER 2001 | 789 REVIEWS The applications of these newly developed tech- nologies are still in their infancy. Because they can now be generated in any region of the genome, marked deletions in mice, like their counterparts in Drosophila, will become invaluable for mapping genetic loci, such as quantitative trait loci 48 . Marked deletions and bal- ancer chromosomes will also continue to gain impor- tance in large-scale, recessive genetic screens in mice and will have a significant impact on efforts to func- tionally annotate the mouse genome. Studying DiGeorge syndrome in mice has illustrated the feasi- bility and benefits of using chromosomal engineering to generate models of human chromosomal rearrangements. Experiments are underway to engi- neer mouse models for other human congenital chro- mosomal disorders, such as Smith?Magenis syndrome (K. Walz and J. Lupski, personal communication) and trisomy 21 (REF. 49). The current mouse models of tri- somy 21 are trisomic for only a portion of mouse chromosome 16, and mutant mice do not show all the � 2001 Macmillan Magazines Ltd 790 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics REVIEWS 47. Liu, P. et al. Requirement for Wnt3 in vertebrate axis formation. Nature Genet. 22, 361?365 (1999). 48. Mackay, T. F. Quantitative trait loci in Drosophila. Nature Rev. Genet. 2, 11?20 (2001). 49. Reeves, R. H., Baxter, L. L. & Richtsmeier, J. T. Too much of a good thing: mechanisms of gene action in Down syndrome. Trends Genet. 17, 83?88 (2001). 50. Sago, H. et al. Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc. Natl Acad. Sci. USA 95, 6256?6261 (1998). 51. Hoess, R. H. & Abremski, K. in Nucleic Acids and Molecular Biology Vol. 4 (eds Eckstein, F. & Lilley, D. M. J.) 99?109 (Springer, Berlin and Heidelberg, 1990). 52. Sadowski, P. D. Site-specific genetic recombination: hops, flips, and flops. FASEB J. 7, 760?767 (1993). Acknowledgements We thank M. Wentland for comments on this manuscript and A. Pao for assistance in preparing the figures. Work in the authors? lab is supported by the National Institutes of Health, the Howard Hughes Medical Institute and the Wellcome Trust. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ AF9 | AML1 | ETO | MLL MGI: http://www.informatics.jax.org/ albino | Hprt | Mll | pink-eyed dilution | Trp53 | Wnt3 OMIM: http://www.ncbi.nlm.nih.gov/Omim/ DiGeorge syndrome | Prader?Willi syndrome | Smith?Magenis syndrome | trisomy 21 FURTHER INFORMATION Cell line Request Form: http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/cell.htm Chromosome 11 deletion map: http://www.mouse-genome.bcm.tmc.edu/ChrEng/deletion.asp Chromosome 11 ENU mutagenesis programme: http://www.mouse-genome.bcm.tmc.edu/ENU/ENUHome.asp Genetic and physical maps of the mouse genome: http://carbon.wi.mit.edu:8000/cgi-bin/mouse/index#genetic NCBI?s mouse genome sequencing page: http://www.ncbi.nlm.nih.gov/genome/seq/MmHome.html STS Physical Map of the Mouse: http://carbon.wi.mit.edu:8000/cgi-bin/mouse/index#phys t(8;21)(q22:q22): http://www.infobiogen.fr/services/chromcancer/Anomalies/ t0821.htm t(9;11)(p22;q23): http://www.infobiogen.fr/services/chromcancer/Anomalies/ t0911.html The Bradley Lab Protocols: http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/ protocol.htm The Sanger Centre: http://www.sanger.ac.uk � 2001 Macmillan Magazines Ltd "
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