Recombinogenic targeting: a new approach to genomic analysis—a review1

Gene 223 (1998) 9–20

Recombinogenic targeting: a new approach to genomic analysis—a review1 Cooduvalli S. Shashikant a,*, Janet L. Carr a, Jaya Bhargava b, Kevin L. Bentley b, Frank H. Ruddle a,c a Department of Molecular, Cellular and Developmental Biology, Yale University, Kline Biology Tower, PO Box 208103, New Haven, CT 06520, USA b Genaissance Pharmaceuticals Inc., 5 Science Park, New Haven, CT 06511, USA c Department of Genetics, Yale University, SHM, PO Box 208005, New Haven, CT 06520, USA Received 13 April 1998; accepted 25 June 1998; Received by J. Wild

Abstract Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of largeinsert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Large-insert cloning vectors; Transgenic analysis; Homologous recombination; In-vivo cloning; pClasper; TAR cloning; BAC; YAC; MAC

1. Introduction Insert size represents one of the most important variables in DNA cloning. One insert size does not fit all needs, and in response to this dictum, a number of cloning systems have been introduced that permit the cloning of inserts of differing sizes. These considerations are particularly relevant with respect to gene mapping. In the 1970s, methods were developed that allowed the cloning of whole chromosomes (approximately * Corresponding author: Tel: 203 432 3516; Fax: 203 432 5890; e-mail: [email protected] 1 Published in conjunction with The Wisconsin Gathering Honoring Waclaw Szybalski on occasion of his 75th year and 20 years of Editorship-in-chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA. Abbreviations: ARS, autonomous replicating sequence; BAC, bacterial artifical chromosome; bp, base pair(s); CEN, centromere; ES, embryonic stem; kb, kilobase(s) or 1000 bp; LCR, locus control region; MAC, mammalian artificial chromosome; Mb, megabase(s) or 1000 kb; oligo, oligodeoxyribonucleotide; ori, origin of DNA replication; PAC, P1 artificial chromosome; PCR, polymerase chain reaction; TAR, transformation associated recombination; YAC, yeast artificial chromosome; YIP, yeast integrated plasmid; YRP, yeast replicating plasmid.

1000 Mb) in an allospecific cell line (Ruddle et al., 1970). This somatic cell hybrid technology facilitated the assignment of genes to a whole chromosome without reference to regional location or linkage order. Cloning of subchromosomal fragments, such as translocation or other rearrangement products, allowed the assignment of genes to subchromosomal regions as small as 1 Mb and provided information on gene order (Ricciuti and Ruddle, 1973). At the time, cloning of genomic DNA in bacteria was not suitable for long-range genomic mapping. Conventional plasmids hold up to 10 kb of DNA, l bacteriophage hold up to 20 kb, and cosmids can hold up to 45 kb (Sambrook et al., 1989). It would take over 30 000 cosmid clones to cover even one average chromosome. The introduction of Yeast Artificial Chromosome ( YAC ) cloning technology provided a means of cloning much larger fragments, from several hundred kilobases to more than 1 Mb. To bridge the gap between cosmids and YACs, bacterial artificial chromosomes and their derivatives, which hold 100–300 kb, were constructed. These vectors all rely on the chance locations of restriction sites, or random shearing of DNA, for determining the boundaries of the clones, which is sufficient for genomic mapping. However, for functional genomics, it is often necessary

0378-1119/98/$ – see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 36 9 - 2

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to clone an exact fragment of DNA for which convenient restriction sites may not be available. In this review, we will briefly summarize properties of large-insert cloning vectors and discuss a new cloning strategy termed ‘recombinogenic targeting’. Recombinational targeting takes advantage of the capacity of S. cerevisiae for faithful recombination between short homologous sequences (homologous recombination). A vector is designed that contains sequences homologous to regions flanking the gene of interest. Homologous recombination between vector and target DNA (recombinogenic targeting) results in a vector containing the insert of interest. This approach permits insert capture and stable propagation of up to 500 kb and the precise determination of insert boundaries. It also has the capability of precise modification of the insert at nucleotide resolution by genetic recombination. We believe that recombinogenic targeting will have many useful applications, ranging from gene discovery to functional analysis of genes in transgenic systems, as we will point out in the sections to follow.

2. Large-insert cloning vectors 2.1. General properties For the purposes of cloning genomic regions, several vectors have been constructed. Salient features of these vectors are listed in Table 1. The first vectors were phage l and cosmids that could hold sequences of 20 and 45 kb, respectively (Sambrook et al., 1989). YACs were developed when the sequences necessary for centromere, telomere, and replication origin (ori) were defined in yeast (Burke et al., 1987). These sequences, along with selectable markers, were combined to create a linear cloning vehicle. YACs can maintain sequences of up to 2000 kb, which was a significant improvement over bacterial and phage vectors that were available at the time. The P1 vector was developed to bridge the gap between YACs and cosmids (Sternberg, 1992). The P1 vector uses a P1 bacteriophage replicon. Clones are generated by ligation followed by packaging in phage, and transfer to bacteria. P1s can hold 70–100 kb, show less chimerism, and, since they are maintained at just one copy per cell, are much more stable than cosmids and YACs. The next step in large insert cloning was the development of bacterial artificial chromosomes (BACs; Shizuya et al., 1992). BACs use the F-factor ori, which, together with par elements, maintains BACs at one or two copies per cell. Because they are directly transformed into bacteria by electroporation rather than packaged through phage, they can hold much larger fragments, up to 300 kb so far. P1 artificial chromosomes (PACs) are similar to BACs in employing electroporation instead

of in-vitro packaging for bacterial transformation, but they rely on the P1 ori, which has the added advantage of a second, multi-copy and inducible ori that enables preparation of large quantities of DNA (Ioannou et al., 1994). Conditional amplification for BAC vectors has also been developed recently (Hradecˇna´ et al., 1998). These three cloning vehicles are comparable to cosmid clones with respect to the ease of handling. Currently, there is considerable interest in building mammalian artificial chromosomes (MACs; Brown et al., 1996; Huxley, 1997). Two different approaches involved in designing MACs can be described as ‘bottom-up’ and ‘top-down’ approaches. In the ‘bottomup’ approach, mammalian DNA already cloned into YACs is used to assemble MACs, and in the ‘top-down’ approach, minichromosomes generated by fragmenting human chromosomes are manipulated and tested for their ability to propagate in mammalian cells. YACs were constructed by conventional recombinant DNA techniques to assemble an ori, a centromere, a selectable marker and two telomeres. However, information on similar mammalian sequences is meager. Recently, firstgeneration human artificial chromosomes have been generated by combining long arrays of a satellite DNA, telomeric DNA and genomic DNA (Harrington et al., 1997). The resulting microchromosomes appear to be stable for several months in cell culture and should provide an opportunity to dissect out those sequences that are necessary for constructing a minimal MAC vehicle. 2.2. Specialized features of large insert-cloning vectors Some cloning vehicles have the ability to positively select for clones with inserts. Cosmids, fosmids, and P1s are packaged by phage, which has a preferred size of packaging ( Kim et al., 1992; Sternberg, 1992). Smaller fragments are packaged less efficiently, leading to enrichment for the larger sequences. Recently, the BAC vector has been modified with the addition of lacZ for color selection of positive inserts (Asakawa et al., 1997). P1 and PAC vectors contain the sacB gene, which allows for direct selection (Ioannou et al., 1994; Perry et al., 1995). The intact sacB gene causes toxic by-products to accumulate in the presence of 5% sucrose, thus selecting against those clones with no insert. There are various features that allow one to map the genomic inserts and array clones into contiguous sets of overlapping clones (contigs). The SP6 and T7 promoter sites allow for end probes to be generated by run-off with SP6 or T7 RNA polymerase. The cosN and loxP sites, which are found in the BAC vector, also allow for the generation of ends for use in restriction mapping by cleavage with l terminase and P1 Cre protein, respectively (Shizuya et al., 1992). Finally the vectorette method for generating end probe does not require any

Low F-factor Stable

Yes No

Low pBR 322; l Unstable (up to 35%) rearranged; Ioannou et al., 1994) Moderate to high????

E. coli

Ligation

Package

Lower efficiency of packaging Yes No

Copy number

Host

Construction by:

Delivery into host

Selection against nonrecombinants

Modify by restriction/ ligation Modify by homologous recombination

aConditionally, multicopy BACs were developed by Hradecˇna´ et al. (1998).

Lower efficiency of packaging

Package

Ligation

E. coli

Single

3/0/0

2/1/0

Unique sites in polylinker (6-bp cutters/8-bp cutters/ mega-nucleases) Chimaerism Replicon Stability

34–45 kb

34–45 kb

Fosmid

Size

Cosmid

Table 1 Plasmid vectors and artificial chromosomes used for genomic cloning

No

Yes

sacB

Package

Ligation

Single; lac inducible lytic cycle to five or six copies E. coli

Low P1 Stable???

2/2/0

70–100 kb

P1

Yes

Color selection (Asakawa et al., 1997) Yes

Electroporation

Ligation

E. coli

Singlea

Low F-factor Stable

4/2/9

To 300 kb

BAC

Yes

Yes

sacB

Electroporation

Ligation

Single; lac inducible lytic cycle to five or six copies E. coli

Low (<3%) P1 Stable

3/1/0

To 300 kb

PAC

Yes

Yes

None

Ligation and/or homologous recombination Electroporation

E. coli/S. cerevisiae

Single

Low F-factor Stable

7/52

To 300 kb*

Clasper

Yes

Yes

Spheroplast

Ligation

E. coli/S. cerevisiae

2–3??

Frequent Yeast Unstable

100 kb to 2 Mb //0??

YAC

Yes

No

Lipofection, cell fusion, or microinjection

Mammalian and avian tissue culture cells Homologous recombination

2 (diploid)

Unknown Mammalian Unknown

Not aplicable

4 Mb—unknown

MAC

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Fig. 1. Methods to alter yeast genome. A black line indicates the yeast chromosome, and a red circle indicates the plasmid. (A) A circular plasmid transformed into yeast is maintained in yeast as a plasmid without integration into yeast genome. (B) Transformation of yeast with an integrating plasmid with or without a double-strand break leads to integration of the plasmid into the yeast chromosome. (C ) Transformation of yeast with exogenously altered gene containing a yeast selectable marker leads to replacement of chromosomal gene by the one-step gene replacement method. (D) Transformation of yeast with integrating plasmid and popping out the vector sequences (single red line) by positive selection leads to integration (double red line) without vector sequences. ( E ) Transformation of yeast with gapped plasmid (red ) containing ends (black) of the gene to recover chromosomal region of same gene by double-strand exchange (see Fig. 2B, bottom drawing).

specific sites to be designed into the vector. This method requires genomic clones to be digested with various restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector-specific primer and a linker-specific primer. This method has been successfully used with YACs and BACs ( Riley et al., 1990; Asakawa et al., 1997). 2.3. Vectors that allow homologous recombination Many of the large-insert cloning vectors discussed above were primarily designed for the purpose of generating contig maps of complex genomes. Although the design of YACs was greeted with enthusiasm for the ability to perform genetic manipulations in yeast, its application towards functional genomics has encountered several problems. A yeast–bacteria shuttle vector, pClasper, was specifically designed for the dual purpose of cloning large-inserts and functional genomics

(Bradshaw et al., 1995). pClasper combines the F-factor ori and a chloramphenicol-resistance gene for propagation and selection in bacteria; the CEN6/ARS4 ori and LEU2 gene for propagation and selection in yeast. Further, it has an extensive polylinker consisting of 12 restriction endonuclease 6- and 8-bp recognition sites for flexibility in cloning particular fragments of interest. The polylinker is flanked on both sides by 18-bp recognition sites for the meganucleases I-PpoI and I-SceI for excision of the intact insert. Like BACs, pClasper can be used for cloning large inserts and to transform bacteria by electroporation, and large amounts of DNA can be easily obtained by conventional methods. In addition, like YACs, pClasper can be maintained in yeast for the purpose of manipulation by homologous recombination. Unlike YACs, pClasper is a circular vector that makes it less vulnerable to rearrangements that are rampant in YAC clones. Two shuttle vectors, ClyA, a derivative of pClasper and pCIRC3, containing P1 ori ( like PACs) were speci-

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Fig. 2. Cloning of exogenous DNA in yeast. (A) Transformation of human or mouse genomic DNA into yeast by in-vitro constructed YACs. (B) Transformation of human mouse genomic DNA along with linear or circular vector into yeast to recover as circular or linear YACs by in-vivo cloning.

fically designed to allow the circularization of YACs into yeast–bacteria shuttle vectors by recombinational cloning (Bradshaw et al., 1995; Frengen et al., 1997). With pCIRC, circularized YACs of up to 220 kb were successfully shuttled to bacteria, but those that were greater than 400 kb were not, suggesting that the apparent size limitation of 300–400 kb seen in BACs and PACs may be due to difficulties in transforming bacteria with such large constructs ( Frengen et al., 1997). Similarly, pPAC-ResQ, a derivative of pClasper, containing two arms of P1/PAC vectors, has recently been used to clone inserts from P1 and PAC clones (J.B., C.S.S., J.L.C., K.L.B., C.T. Amemiya and F.H.R., unpublished observations). Further extension of this principle of recombination-targeted cloning includes the derivation of linear and circular YACs containing human genomic DNA (see Section 4.4). All the vectors discussed above use homologous recombination within yeast. However, recently, methods to modify BACs by homologous recombination in bacteria have been reported ( Yang et al., 1997). The yeast–bacteria shuttle vectors described above, in particular pClasper, have the potential to be used for conventional contig analysis. The similarity of pClasper with BAC and YAC vectors suggests that large inserts can be cloned and maintained in both bacteria and yeast. Though pClasper does not contain T7 and SP6

promoter sequences or the cosN and loxP sites for the generation of riboprobe end-probes, it can be used for contig analysis by using the vectorette method of generating end probes, which does not require specific promoter sequences ( Kim et al., 1992).

3. Recombinational-targeted cloning methods Genetic recombination involves the exchange of DNA sequences between two DNA molecules [for reviews, see Kucherlapati and Smith (1988)]. There are three major types of recombination: homologous, site-specific and illegitimate. In homologous recombination, exchange occurs between two DNA molecules sharing regions of extensive sequence homology. In site-specific recombination, exchange occurs at specific sites that are targets of specific recombinases. In illegitimate recombination, the exchange occurs at sites that are neither specific nor homologous. Genetic recombination is common to all organisms and contributes towards the generation of genetic diversity. The recombinational machinery of several experimental organisms has been successfully used for modification of endogenous genes, for example, knocking-out genes in various organisms including mice. In the following section, we will limit our discussion primarily to the use of homologous recombination-

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based methods developed in yeast for cloning and modification of genes. Transposon-based methods are excluded from the scope of this review. 3.1. Homologous recombination in yeast Yeast are more efficient at homologous recombination than other organisms. Transformed DNA ( lacking in autonomous replicating sequences, ARS) almost exclusively integrates into the yeast genome by homologous recombination (Hinnen et al., 1978). This high frequency of homologous recombination allows one to easily introduce changes in the yeast genome (Fig. 1). Two popular methods to manipulate yeast genome by this approach are so called pop-in/pop-out and one-step gene replacement methods (Scherer and Davis, 1979; Rothstein, 1983). In the pop-in/pop-out method, a gene of interest is specifically altered in a yeast integration plasmid ( YIP) containing URA3, which can be selected both positively and negatively (Orr-Weaver et al., 1981). This plasmid is then transformed into yeast and selected for URA3. This results in the integration of the YIP at the homologous locus, creating a duplication of the sequences introduced at the site (pop-in). The frequency of homologous recombination is greatly enhanced by introducing a double-stranded break in the transforming YIP. In the next step, yeast are selected against URA3 by plating on media containing 5-fluoro orotic acid (5-FOA). As a result, the YIP sequences are excised from the yeast genome (pop-out) via homologous recombination between the two tandem copies of the gene. In half of the events, the mutated gene will be excised out, and in the other half, mutation stays intact on the chromosome, depending upon where the recombination takes place. This pop-in/pop-out strategy is the most effective strategy for introducing specific mutations into a gene without affecting the rest of the locus. In the one-step gene replacement method, an altered DNA fragment with a selectable marker is transformed into yeast in the absence of the vector sequence (Rothstein, 1983). This method is used either to introduce a marker gene at the locus or to introduce a deletion into a given locus. A selectable marker can be introduced either in the coding region or in the 3∞ untranslated region without disrupting the gene. Deletions can be constructed by appropriately selecting homologous DNA fragments that flank a selectable marker such as LEU2. The replacement construct is then excised from the vector sequence and transformed into yeast. This leads to the replacement of wild-type sequences with in-vitro manipulated constructs. Transformation of yeast with plasmids containing ARS and CEN sequences results in stable maintenance of the plasmid without integration. Yeast replicating plasmids ( YRP) have been extensively used for cloning

genes by complementation (Beggs, 1978). When doublestranded breaks were introduced into a YRP and transformed, it was noticed that the gap was filled with a copy of the homologous fragment from the yeast genome (Nasmyth and Reed, 1980; Orr-Weaver et al., 1981). This non-reciprocal transfer of information, called gaprepair, can be used to recover yeast chromosomal alleles. This is done by transforming yeast with a gapped YRP containing the appropriate recombinogenic arms. The missing fragment will then be repaired off the chromosomal locus by gap repair. This principle of gap repair has been employed to clone and modify exogenous DNA in yeast (Fig. 2). DNA fragments from YACs and other vectors can be rescued into gapped yeast–bacteria shuttle vectors such as pClasper that contain appropriate homologous arms on either side of the gap (Bradshaw et al., 1995). Adenoviral and human genomic DNA fragments have been recovered as YACs by transforming yeast with the genomic DNA of interest along with two arms of YACs flanked by homologous arms derived from genomic DNA ( Ketner et al., 1994; Larionov et al., 1994a, 1996a,b, 1997). Applications of these methods towards functional genomic analysis are discussed in the following sections. 3.2. Homologous recombination in bacteria E. coli has an efficient and well-characterized homologous recombination system [reviewed in Kucherlapati and Smith (1988)]. However, methods for recombinational cloning in E. coli have not been as extensively used as in yeast. Based on the observation that E. coli also has a gap-repair system, methods to clone DNA fragments by transforming with gapped plasmids containing homologous arms have been developed (Bubeck et al., 1993; Oliner et al., 1993). Methods similar to one-step gene replacement in yeast have been employed to construct recombinant adenovirus (Chartier et al., 1996). In this method, adenoviral genomic DNA is cloned by cotransforming adenoviral genome and a gapped plasmid containing homologous arms into E. coli. Similarly, a two-step gene replacement method has been used to construct recombinant adenoviral genomes (Crouzet et al., 1997). This method involves formation of a co-integrate and subsequent resolution of it resulting in recombinant molecules as in the case of the popin/pop-out method. Transformation of E. coli with a linear DNA fragment flanked by Chi sites (which stimulate recombination) has been shown to result in integration of DNA by homologous recombination (Dabert and Smith, 1997; Jessen et al., 1998). This method can be used for gene targeting in E. coli and other bacteria. A gene replacement method for the modification of BACs has been described previously by Yang et al. (1997). In this method, recombination-deficient bacteria

C.S. Shashikant et al. / Gene 223 (1998) 9–20

are transformed with a temperature-sensitive vector containing the recA gene and the replacement construct. At the permissive temperature, recombination leads to the co-integrate formation. Resolution of the co-integrate leads to the recombination product with the loss of RecA function. Resolution can lead to the formation of original and recombinant BACs. This method has a wide potential for modifying large inserts for functional analysis.

4. Applications of recombination-targeting methods 4.1. Transgenic mice as a model for functional analysis The recombination-targeting approach to cloning is likely to enhance functional genomic analysis using the transgenic mouse as a model system. The advantages of mouse models for the study of mammalian physiology; human diseases, and developmental abnormalities have been extensively reviewed (Jaenisch, 1988; Hanahan, 1990; Winter, 1996; Rubin and Smith, 1997; Bedell et al., 1997a,b). Despite its intrauterine development and other features that make mice a ‘low-throughput reagent’, the mouse is foreseen as a leading system for most functional genomic studies (Rubin and Smith, 1997). Transgenic mouse technology developed in the early 1980s has led to the creation of mice carrying various genes associated with human diseases, for example, oncogenes, and has provided evidence for the etiology of pathogenesis (Bedell et al., 1997a,b). Limitations of conventional transgenic technology are due to the limited size of transgenic constructs prepared with DNA fragments cloned in pUC or pBR322 based plasmid. Many of the small transgenic constructs derived from cDNA clones express poorly in the absence of intronic sequences and are vulnerable to the position effect of local enhancers present at the site of genomic integration (Jaenisch, 1988). Regulatory sequences are often scattered through 5∞, 3∞ and intronic regions and at large distances, thus eluding identification. Thus, with transgenic mice generated using limited size constructs, it is difficult to create models that resemble every aspect of the gene expression, which is often critical in our understanding of the gene function. 4.2. Large-insert transgenics Many of the above-mentioned shortcomings have been resolved with the development of large-insert cloning vehicles, especially YACs. YACs can be introduced into mice either by direct pronuclear injection or by ES cell technology (Lamb and Gearhart, 1995; Peterson et al., 1997). In the latter method, yeast spheroplasts containing YACs are fused with ES cells, or YACs are introduced into ES cells by lipofection. ES cells carrying

15

YAC DNA are then injected into mouse blastocysts to create chimeric mice. Many applications stemming from YAC transgenics have been reviewed elsewhere (Lamb and Gearhart, 1995; Peterson et al., 1997; Rubin and Smith, 1997). Briefly, YAC transgenics have so far made it possible to examine large genes and gene complexes; to identify long-acting cis-regulatory elements; to create transgenic models that more faithfully reproduce the functional aspects of endogenous loci; to complement known genetic mutations; and to study higher-order chromosomal structures such as X-chromosome inactivation (Lamb and Gearhart, 1995; Peterson et al., 1997; Rubin and Smith, 1997). These approaches have emboldened investigators to create ‘mouse in vivo libraries’ to examine complex traits (Rubin and Smith, 1997). For example, Smith et al. (1995, 1997) have established transgenic mouse panels, each containing distinct overlapping YACs that together span about 2 Mb of human chromosome 21q22.2, a region of significance for the analysis of various abnormalities associated with Down’s syndrome (Smith et al., 1995, 1997). 4.3. Modification by recombinational targeting for transgenic analysis Recombinational cloning has so far led to relatively few applications for transgenic analysis. Further refinement of functional analysis of genes will require modifications of large inserts. Precise alterations in both the coding and regulatory sequences are essential to understand the role of specific sequences in gene function. The development of YACs was instrumental for making the powerful homologous recombination system of yeast available for such modifications. A few examples of modification of regulatory sequences include utilization of the pop-in/pop-out method for introducing mutations into a b-globin locus YAC (Bungert et al., 1995; Peterson et al., 1995, 1996). A point mutation introduced in the distal CCAAT box of the A globin gene c resulted in the delayed c–b-globin gene switch and persistence of c-globin expression, characteristic of the Greek A form of the hereditary persistence of fetal c hemoglobin (HPFH; Peterson et al., 1995). Similarly, alterations in the DNase I hypersensitive regions ( HSs) flanking the b-globin gene were engineered by the popin/pop-out method (Peterson et al., 1996). Transgenic analysis of mutated b-globin YACs revealed altered levels of expression of globin genes, suggesting that synergetic interactions at HSs of the locus control region (LCR) control the switching of gene expression at this locus (Peterson et al., 1996). One immediate application of homologous recombination for the analysis of regulatory regions is the generation of reporter gene constructs likely to contain all the cis-acting elements of the gene. Conventional

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reporter gene analyses have examined sequences immediately surrounding the gene for elements capable of conferring the expression pattern of the endogenous gene on a reporter, usually the lacZ gene. However, most studies have succeeded in recapitulating only certain aspects of complex expression pattern of the endogenous gene. This is presumably due to missing regulatory sequences, either distant sequences not contained with the cloned region or proximal sequences often eliminated for conveniences of the in-vitro subcloning procedures. Few studies have taken advantage of homologous recombination to generate reporter gene constructs. In the study of Hoxc8 gene regulation, pClasper was used to rescue a 27-kb DNA fragment surrounding Hoxc8 from a YAC clone, and a lacZ–URA3 cassette was then inserted in the coding frame of the sequence by the onestep replacement method (Bradshaw et al., 1995, 1996). In another study, a 29-kb DNA fragment surrounding a lamprey HoxQ8 gene was cloned into a pClasper by in-vitro subcloning, and the lacZ–URA3 cassette was similarly inserted by the one-step replacement method (Carr et al., 1998). Cis-acting elements capable of directing region-specific expression in transgenic embryos were detected with constructs generated by recombinational cloning. Using homologous recombination in bacteria, an IRES–lacZ gene was introduced into a 130-kb BAC containing the mouse zinc finger gene, RU49 ( Yang et al., 1997). In these cases, long-range cis-regulatory elements, not identifiable with conventional transgenic constructs, were detected. An emerging consensus in the study of regulation of genes is that if smaller transgenic constructs fail to identify regulatory elements within the proximal regions of the gene, it is desirable to analyze as much as 100-kb DNA surrounding the gene, which includes all exons and introns and additionally 10–20-kb fragments both upstream and downstream of the gene. Such a genomic fragment can be identified by screening either existing P1, BAC, PAC or YAC genomic libraries. Either bacteria or yeast genetic systems can be employed to introduce reporter genes. Although methods for homologous recombination in bacteria have been demonstrated with BACs, other bacterial cloning vehicles can be similarly targeted. The inserts cloned into pClasper can be modified using both bacterial and yeast recombination systems. Inserts from standard bacterial vectors can also be captured into pClasper for the purpose of modification by recombination in yeast. Recombination-targeted cloning has thus made longrange cis-regulatory analysis more feasible. These simple methods of introducing reporter gene or altering specific sequences should not only identify cis-acting elements, but also enable more difficult and challenging experiments. This includes elucidating interactions at multiple cis-acting elements, interactions between global regulatory sequences and gene-specific enhancers, and sharing

of enhancers between two or more closely linked genes. These applications can be discussed in the context of the regulation of Hox genes ( Krumlauf, 1994; Lufkin, 1996). Conventional transgenic analyses have identified multiple enhancers located either in 5∞, 3∞ or intronic regions of Hox genes that are capable of recapitulating many, but not all, aspects of complex patterns of expressions ( Krumlauf, 1994; Lufkin, 1996). For some genes, the regulatory elements have not been identified, despite examining entire intergenic sequences, suggesting that these elements may be located farther away. In a few instances, the same regulatory region appears to be shared between neighboring genes ( Van der Hoeven et al., 1996; Gould et al., 1997). Further, the clustered organization of Hox genes has several striking features ( Krumlauf, 1994; Ruddle et al., 1994). Genes within the cluster exhibit a colinear relationship with respect to the anterior boundary of expression along embryonic axes (spatial colinearity), the time of activation during ontogeny (temporal colinearity), and response to the morphogen retinoic acid (colinearity of sensitivity). Many aspects of the clustered organization are highly conserved among metazoans (Ruddle et al., 1994). The functional basis of the structural organization of Hox gene clusters can be systematically analyzed using the recombination-targeting approach. DNA fragments (130–200-kb) containing an entire Hox cluster can be isolated in pClasper (Bradshaw et al., 1995). Individual genes can then be inactivated and replaced with reporter genes, gene-specific elements (e.g. retinoic-acid-sensitive elements) can be specifically altered and their effect on the expression of each gene in the cluster can be examined. Other applications of recombination targeting include making targeting constructs for knock-out and knock-in experiments (Storck et al., 1996). Although most of these targeting constructs are currently generated by in-vitro sub-cloning procedures, recombination targeting should greatly increase the ease with which such large constructs are generated. For this purpose, construction of a pClasper library of strain 129 mouse DNA would further simplify the production of targeting constructs. Recombination-targeted cloning should also help in designing more sophisticated ‘domain-swapping’ and amino-acid substitutions for the purpose of assessing which protein-coding regions are critical for function. 4.4. Recombination-targeted cloning of genes directly from genomic DNA The possibility of using recombination targeting to clone genes directly from genomic DNA without going through screening libraries came from several independent groups (Spencer et al., 1993; Larionov et al., 1994b; Bradshaw et al., 1995). The human adenovirus type 2 (Ad2) genome was converted into a YAC by cotrans-

C.S. Shashikant et al. / Gene 223 (1998) 9–20

forming Ad2 DNA with two YAC arms, each containing a recombinogenic fragment derived from the right or left arm of the Ad2 DNA ( Ketner et al., 1994). The adenovirus genome was recovered as a YAC, even when it was diluted with genomic DNA, suggesting that it was possible to recover even single copy genes by recombination targeting. Larionov’s group demonstrated recombination between two YACs co-penetrating the same yeast cell as an important source for chimerism observed in many YAC libraries ( Kouprina et al., 1994; Larionov et al., 1994b). They proposed that common repeat sequences, such as Alu repeats, provide homologous sequences for recombination. Based on these observations, a recombinationtargeting method was devised, referred to as transformation-associated recombination ( TAR), to capture human DNA sequences as either linear or circular YACs (Larionov et al., 1996a,b, 1997). Using Alu sequences as recombinogenic ends in targeting vectors, YACs containing human DNA ranging from 70 to 600 kb were produced. Using Alu sequences in combination with sequences found specifically in rDNA genes, YACs were obtained that contained human rDNA genes ( Kouprina et al., 1997). Similarly, using mouse repetitive sequences as recombinogenic ends, mouse DNA has been cloned as YACs (Cancilla et al., 1998a). Further, even single copy genes could be recovered as YACs using recombinogenic ends from flanking regions of the gene (Larionov et al., 1997; Kouprina et al., 1998). For example, by co-transforming human DNA with a targeting vector that contained recombinogenic ends for the BRCA2 gene, one in the 5∞ promoter region and the other in the last exon of the gene, it was possible to isolate the intervening 90 kb of genomic sequences representing the entire coding sequence (Larionov et al., 1997). The cloned YAC was then converted to a BAC by retrofitting it with mini-F sequences resulting in a vector that was similar to pClasper. These studies have clearly demonstrated that the recombination-targeting approach can be used for the direct isolation of uncloned genes or DNA segments. A number of applications of recombination-targeted cloning can be envisaged. Using human-specific repetitive DNA as the recombinogenic end, it is possible to selectively isolate human DNA, such as specific human chromosomal fragments from rodent–human hybrid cell lines or specific chromosomal regions (Larionov et al., 1996a,b; Cancilla et al., 1998b). A second major application of the ability to clone specific chromosomal genes, families of genes and single-copy genes by recombination-targeted cloning is for comparative functional genomic studies. With the discovery of many conserved gene families among diverse organisms, there has been an increased interest in understanding evolutionary relationships among species by genomic analysis (Ruddle, 1997). Homologous genes and regulatory sequences

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isolated from different species are often tested for function in experimental organisms such as mouse and Drosophila. Many homologous sequences are often isolated by PCR using degenerate primers designed from conserved motifs. These methods often yield small DNA regions for further analysis. Recombination targeting, using conserved sequences such as the homeobox sequences as recombinogenic ends may aid in the isolation of surrounding sequences such as entire Hox clusters. Another significant application of recombinationtargeted cloning will be towards the screening of the human population for disease genes. There has been a rapid explosion in the number of identified humandisease genes and their corresponding pathogenic mutations (Cooper and Krawczak, 1996; Krawczak and Cooper, 1997; Cooper et al., 1998). Many of these disease genes are not only large in size, but also have a wide spectrum of mutations (Beroud et al., 1996; Gottlieb et al., 1996; Hoang et al., 1996; Beroud and Soussi, 1997; Cariello et al., 1997). These mutations may affect several aspects of gene function, including transcriptional and translational regulation and activity of the protein. Techniques such as comparative hybridization and DNA chip arrays have facilitated simultaneous analysis of large genomes from different individuals ( Kallioniemi et al., 1992; DeRisi et al., 1996). These studies will be greatly enhanced if the defective locus can be rapidly isolated from a large population. Recombination-targeted cloning methods should allow the rapid isolation of disease gene loci as YACs using human DNA samples from a large number of individuals by simply cotransforming with a targeting vector containing the appropriate recombinogenic ends. Genes thus isolated can be studied for mutations in the protein coding region using a number of available mutation detection techniques (Gibbs and Caskey, 1987; Cotton et al., 1988; Orita et al., 1989; Abrams and Stanton, 1992; Kallioniemi et al., 1992; Ravnik-Glavac et al., 1994; DeRisi et al., 1996). Further, functional assays can be designed to detect mutations in the regulatory regions in a variety of expression systems, including yeast, mammalian cell culture and transgenic mice.

5. Conclusions The field of functional genomics is currently witnessing a number of innovations geared towards rapid analyses of genetic loci. Three developments discussed in this review include large-insert cloning vectors, recombination-targeted cloning and transgenic mouse-models. Large-insert vectors discussed here are of three types: bacterial, yeast and yeast–bacteria shuttle vectors. Bacterial cloning vectors such as BACs, PACs and P1s

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provide a distinct advantage in terms of ease of handling and lesser degree of chimerism observed among clones as compared to YACs. YACs provide a larger cloning capacity and ease of manipulation using the yeast genetic system. However, YACs have significant drawbacks in terms of stability and chimerism. It is estimated that 40–60% of clones from some human and mouse YAC libraries consist of chimeric clones (Monaco and Larin, 1994). Further, YACs have a low cloning efficiency. Separation of YAC DNA poses difficulties as YACs are often in the same size range as yeast chromosomes. Yeast–bacteria shuttle vectors, especially pClasper, combine many desirable features of both yeast and bacterial cloning vectors. The inserts cloned in pClasper can be stably maintained in yeast in the circular form, which is less prone to chimerism compared to linear YACs. They can be genetically modified in yeast and then shuttled to bacteria for the purpose of preparation of large quantities of DNA for functional analyses. pClasper can also be used for genomic library construction and conventional contig analysis. Its extensive polylinker provides more flexibility for direct in-vitro cloning of DNA fragments. Recombination-targeted methods have been traditionally developed in yeast and, only recently are techniques in bacteria emerging. These methods will enhance the ability to clone and modify large inserts for functional analysis. This, in turn, will allow us to assay the genome more rapidly, using the transgenic mouse model, contributing greatly to the study of gene function relevant to growth, development, disease and disorders.

6. Dedication Dedication to Waclaw Szybalski (by Frank H. Ruddle): I first met Waclaw Szybalski in 1958 at a scientific meeting in San Francisco dealing with the biological effects of ionizing radiation. I was then a graduate student in U.C. Berkeley and reported on my use of X-rays to introduce cytogenetic markers into tissue culture cell populations. Waclaw was interested in my results and, in the course of our discussion, asked directions to Berkeley where he was scheduled to meet with Gunther Stent, one of my lecturers. Having no car, I guided Waclaw to the bus and we continued to talk. I told him of my ambition to develop ‘somatic cell genetics’ using cultured somatic cells, and he responded with a fully worked out scheme for clonal selection based on drug resistance mutations and their use in complementation systems. In fact, he even recommended the use of mutations involving hypoxanthine–guanine phosphoribosyl transferase. I was greatly impressed and receptive since my mentor at Berkeley, Morgan Harris, was doing pioneeering work on drug resistance in chick cell culture at that time. Several years later, drug-resistance systems came into vogue and played a critical role in the

development of somatic cell genetics systems and in the use of cell hybrids in gene mapping. Waclaw went on to make original and seminal contributions to transformation of mammalian cells (Szybalska and Szybalski, 1962; Scangos and Ruddle, 1981). My chance meeting with Waclaw was a big boost to my scientific career not only in terms of the information conveyed, but very much on a personal level, being taken seriously as a scientific colleague by an established senior scientist. He has continued to be a revered colleague and a good friend over the years. It is with genuine pleasure that I dedicate this article (still dealing with a somatic cell genetics topic) to Waclaw some 40 years after our first encounter.

Acknowledgement This work is supported by NIH grant GM09966 to F.H.R.

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