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Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat,Rattus norvegicus
GENOMICS
39, 385– 392 (1997) GE964494
ARTICLE NO.
Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat, Rattus norvegicus LI CAI,* LEONARD C. SCHALKWYK,† ANDREINA SCHOEBERLEIN-STEHLI,‡ ROBERT Y. L. ZEE,* AVRIAL SMITH,† THOMAS HAAF,† MICHEL GEORGES,‡ HANS LEHRACH,† AND KLAUS LINDPAINTNER*,§,1 *Cardiovascular Division, Brigham and Women’s Hospital, Department of Cardiology, Children’s Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; †Max Planck Institute for Molecular Genetics, Berlin– Dahlem, Germany; ‡Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Lie´ge, Lie´ge, Belgium; and §Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany Received August 23, 1996; accepted October 28, 1996
Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20– 30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. q 1997 Academic Press
INTRODUCTION
The laboratory rat, Rattus norvegicus, is one of the most widely studied experimental organisms in biomedical research. The rat’s size — large enough to allow complex physiological measurements, yet small enough 1 To whom correspondence should be addressed at Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis Street, Thorn 1103, Boston, MA 02115. Telephone: (617) 732-8173. Fax: (617) 2646830. E-mail: [email protected].
for economical breeding— and the availability of a large number of inbred strains have made it a favored animal in physiological research (Hedrich, 1990). More recently, the rat has been recognized as an important experimental system for genetic studies of multifactorial disease, such as hypertension, stroke, diabetes, and renal failure. These strains represent a useful adjuvant to the study of complex disease in humans, as they preserve the polygenic character of the disorder, but allow a reduction of complexity provided by the lack of genetic heterogeneity, by standardization of environmental influences, and by the imposition of programmed breeding schemes. Thus, the feasibility of mapping quantitative trait loci (QTL) in mammals was first demonstrated in cross-breeding experiments with inbred strains of rats (Hilbert et al., 1991; Jacob et al., 1991). However, the lack of genomic resources for the rat —as compared to human or mouse— has significantly impeded progress toward identification of traitrelevant genes. The availability of polymorphic markers — while continuously improving — is still scarce, and the map of the rat genome remains rather sketchy (Levan et al., 1991; Serikawa et al., 1992; Yamada et al., 1994; Jacob et al., 1995; Pravenec et al., 1996; http:// www.RATMAP). To utilize fully the emerging panels of more densely spaced polymorphic markers, as well as to aid in the development of targeted markers, a set of large-insert genomic libraries (yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), cosmids) is an essential requirement. Here, we report the establishment and characterization of the first YAC library for the rat. MATERIALS AND METHODS
1. Construction of a Rat YAC Library (a) Preparation of high-molecular-weight DNA. A fresh spleen was taken from a stroke-prone, spontaneously hypertensive rat (a
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fully inbred strain commonly used in cardiovascular research) and cut in half. Cells were extruded by blunt manipulation into a petri dish containing 20 ml ice-cold phosphate-buffered saline (PBS: 1% NaCl, 0.025% KCl, 0.0143% Na2HPO4 , 0.0025% KH2PO4). Spleen cells were suspended in 50 ml PBS, and coarse tissue debris was allowed to settle by gravity for 5 min. The supernatant cell suspension was transferred to a new 50-ml Falcon tube and cells were pelleted by centrifugation at 600g for 10 min at 47C. The pellet was resuspended by gentle agitation in 50 ml cold PBS and counted using a hemocytometer. Nucleated cell number was estimated by multiplying the raw data by a factor of 0.6 as an empirically found adjustment to account for the presence of erythrocytes. Cells were re-pelleted by centrifugation as described and resuspended in PBS to yield a concentration of 4 1 107 cells/ml. This cell suspension was mixed with an equal volume of 1.5% low-melting-point agarose (dissolved in PBS) at 407C and cast as 0.75% agarose plugs into 100-ml molds (Bio-Rad). Plugs were equilibrated in ESP solution (0.5 M EDTA, 1% N-lauroylsarcosine sodium, 1 mg/ml proteinase K) and incubated at 507C for 48 h with gentle shaking. Plugs were subsequently washed against TE (10 mM Tris – HCl, 1 mM EDTA, pH 8.0) at 507C for 30 min, then against 1 mM PMSF in TE at 507C for 30 min, and finally against TE at room temperature. Plugs were stored in 0.5 M EDTA at 47C until use, when they were again equilibrated against 11 TE. (b) YAC vector preparation. The 19.5-kb vector, pCGS966 (19.5 kb, Smith et al., 1990), transformed into Escherichia coli strain XL1blue [recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac (F* proAB lacIq lacZDM15 Tn10], was used for construction of the library. Transformed cultures were grown overnight at 377C on LB agar plates containing 50 mg/ml ampicillin. Four single colonies were picked, and plasmid DNA was prepared and digested with HindIII for agarose-gel verification that no telomeric deletions had occurred during propagation in E. coli. Vector DNA was prepared using CsCl gradient centrifugation (Sambrook et al., 1989). For cloning, the vector was digested to completion with EcoRI and BamHI, as verified by agarose gel electrophoresis. DNA was dephosphorylated at 377C for 40 min using calf intestinal phosphatase (Boehringer Mannheim). Digestion products were purified using StrataClean Resin (Stratagene), precipitated, and dissolved in TE. Completeness of dephosphorylation was verified by the absence of a ligation product on agarose gel electrophoresis after overnight incubation of 200 ng of dephosphorylated DNA in the presence of DNA ligase, ATP, and Mg/ at 167C. Additional quality controls were carried out by transforming unrestricted vector, restricted vector, and restricted, phophatased, rekinased, and religated vector into E. coli. (c) Partial digestion of genomic DNA. Partially digested genomic DNA was produced by concomitant incubation with both the restriction endonuclease, EcoRI (NewEngland BioLab), and the corresponding EcoRI methylase (NEB). One agarose plug (containing about 12 mg genomic DNA) was equilibrated against 400 ml EcoRI digestion mix (80 mM S-adenosylmethionine, 2 mM MgCl2 , 100 mM NaCl, 50 mM Tris –HCl, pH 7.5, 1 mM DTT, 0.5 mg/ml BSA, 4 mM spermidine, and variable amounts of EcoRI/EcoRI methylase) on ice for 30 min. The partial digestion reaction was started by transferring the tubes to a 377C water bath for 4 h and was stopped by adding 30 ml 0.5 M EDTA (pH 8.0). The ratio of EcoRI to EcoRI methylase was adjusted (EcoRI/EcoRI methylase: 1/60– 1/70) to yield a majority of DNA fragments in the size range above 500 kb. (d) First size selection. The partially digested DNA was electrophoretically size-fractionated on a 1% low-melting agarose gel (FMC, Seaplaque GTG) using a CHEF apparatus (Bio-Rad). Megabase-size DNA was separated in 0.51 TBE buffer (11 TBE is 0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8.0) at 117C, 6 V/cm for 16 h, with a 25-s switch time, resulting in the concentration of all DNA fragments larger than 500 kb in the compression zone. The compression band was excised for further manipulation. (e) Ligation. The gel piece containing the compression band was dialyzed three times for 30 min each against ice-cold 11 ligation buffer (50 mM Tris –HCl, pH 7.5, 10 mM MgCl2 , 30 mM NaCl, 0.75 mM spermidine, 0.3 mM spermine). Vector DNA prepared as detailed
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above was then added in approximately 20 molar excess. The sample was heated to 687C for 10 min, mixed by gentle inversion, and incubated at 377C for 1 h to allow for uniform distribution of vector and genomic DNA. The ligation reaction was started by adding DTT (1 mM ), ATP (10 mM ), and 200 units of ligase per milliliter (NEB), followed by gentle inversion of the tube, and the reaction was completed by overnight incubation at room temperature. (f) Second size selection. The solidified low-melting agarose gel block containing the ligation product was cut into 2-mm-thick slices that were equilibrated against ice-cold 0.51 TBE with two changes of buffer, for 30 min each. Slices were then loaded on a 1% lowmelting agarose gel and size selection was carried out analogously to the first size selection. The compression band was excised equilibrated against ice-cold TENP buffer (10 mM Tris– HCl, pH 7.5, 1 mM EDTA, 30 mM NaCl, 0.75 mM spermidine, 0.3 mM spermine) three times, for 30 min each. To prepare the DNA sample for transformation, the gel slices were melted at 687C for 10 min, and equilibrated at 407C for 10 min, and 5 units of beta-agarase I (NEB) was added for each 1 ml of gel volume, with a subsequent incubation at 407C for 2 h. (g) Transformation. We modified the protocol communicated by Burgers and Percival (1987), using zymolyase (20T, ICN), at 8– 22 units per 50 ml of yeast cells grown to an OD600 of 4.0 instead of lyticase. Likewise, SPEM (1 M sorbitol, 1.6 mM NaH2PO4, 8.4 mM Na2HPO4, 5 mM EDTA. 0.2% b-mercaptoethanol) was used instead of SCEM (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, 30 mM b-mercaptoethanol). Transformants were selected on uracildeficient medium and were visible after 2 –3 days. (h) Storage of the YAC library. Single colonies were transferred manually with sterile toothpicks into 96-well microtiter plates containing 180 ml SD medium [2% glucose, 0.072% CSM -trp -ura, (BIO101), 0.67% yeast nitrogen base without amino acids, (DIFCO)] and were grown for 3 days at 307C. As a consequence of the more stringent double-selective medium (-trp, -ura), about 5– 15% of wells failed to show growth. These empty wells were recorded. After mixing in an additional 180 ml storage medium (YPD with 40% glycerol; YPD is 1% yeast extract, 2% bactopeptone, 2% glucose) using a 12channel pipette, three master-aliquots of 100 ml each were prepared and frozen at 0807C. A single replica plate made using a 96-pin replicator was likewise allowed to grow and was split into three aliquots containing 100 ml each.
2. Characterization of the YAC Library We used a combination of several approaches to determine qualitative and quantitative characteristics of the library, including measurements of average insert size, screening of the library by PCR using pools and by Southern hybridization using high-density spotted filters, and fluorescence in situ hybridizaton on karyograms to determine the fraction of chimeric clones. (a) Average insert size determination. Randomly selected YAC clones were grown in 5 ml SD medium for 3 days at 307C in a shaking incubator. Two 100-ml agarose plugs were made from the culture as previously described (Sambrook et al., 1989). Half of a plug was placed into the sample slot of a pulsed-field gel and run for 18 h at 6 V/cm, 117C, and with a switch time of 60 s. After depurination with 0.2 M HCl for 30 min the gel was blotted against a nylon membrane (Hybond-N/, Amersham). Total rat genomic DNA, sheared and radiolabeled with [a-32 P]dCTP using Klenow polymerase in the presence of random hexamer primers, was used as a probe to detect the inserts. A standard yeast chromosome marker was included to determine the insert size. (b) Preparation of PCR screening pools. A 3-dimensional pooling strategy was used to prepare DNA pools for PCR screening. Three copies of the YAC library (replicas from one of the six master copies) were grown in 96-well microtiter plates containing 200 ml SD medium per well, at 307C for 3 days. Eight 96-well microtiter plates were grouped as a block and colonies were pooled using an 8- and a 12channel manifold vacuum device (Sigma) to combine wells from one
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RAT YAC LIBRARY row (x/y plane, yielding 8 row pools) or one column (y/z plane, yielding 12 column pools) from all 8 plates, or wells from one entire plate (x/ z plane, 8 plate pools), providing a total of 28 primary pools from each block for unequivocal addressing of a single well among all 768 contained within the block. DNA was extracted using a previously described method (Sambrook et al., 1989). In brief, pooled cell suspensions (24 ml from plate and row pools and 16 ml from column pools) were centrifuged at 600g for 5 min to pellet yeast cells. Cell pellets were resuspended in 5 ml 50 mM EDTA (pH 8.0) and pelleted as before. The supernatant was discarded, and the cell pellet was dried at room temperature for 10 min and then resuspended in 200 ml lyticase solution (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, pH 8.0, 5% b-mecaptoethanol, and 80 units of lyticase). Following a 20-min incubation at room temperature, 200 ml of 2% lowmelting-point agarose in 125 mM EDTA, pH 8.0, was added, mixed with the cell lysate by gentle agitation, and allowed to solidify at room temperature for 20 min. The resulting agarose plugs were suspended in 2.5 ml 50 mM EDTA, pH 8.0, containing 5% b-mercaptoethanol, and incubated at 307C overnight. After removal of this solution, the agarose plugs were incubated in 2.5 ml ESP solution (450 mM EDTA, pH 8.0, 1% SDS, 1 mg/ml proteinase K) at 507C for 36 h and then washed once with TE, containing 1 mM PMSF, and twice with plain TE. The plugs were melted at 407C after adding 600 ml TE and finally incubated with agarase (Sigma). Secondary (‘‘super’’) pools were generated by pooling equal portions of all 28 primary pool DNA samples from each block of 8 plates. A total of 67 secondary pools were thus generated from 1875 primary pools, representing 535 96-well YAC plates. All primary and secondary pools were stored in deep-well 96-titer plates (Beckman) at 0807C. Initial quality assessment of the YAC pools was carried out by interrepetitive sequences (IRS)-PCR using primers derived from the rat ID element, revealing a characteristic smear from the secondary DNA pools and several distinct bands from the primary DNA pools. Subsequent screening with locus-specific primer pairs was conducted according to a two-stage approach by first amplifying from secondary pools and then determining the specific clone address by subsequent PCR using the respective set of 28 primary pools. (c) Preparation of high-density grid-spotted filters. To allow the use of high-throughput robotic processing, we first generated a copy of the library in 384-well format by replicating four 96-well YAC plates each into one 384-well microtiter plate (Genetix) filled with (ura) SD medium using a 96-pin replicator (Genetix). This spotting copy was allowed to grow for 3 days at 307C. A 4 1 4 duplicate pattern was used for generation of the high-density filters on 22 1 22 cm nylon membranes (Hybond-N/, Amersham). Each filter provided 6 fields corresponding in size to the footprint of one 384-well plate; the area of each single well was represented on the filter by a 4 1 4 array of 16 dots. By spotting each well as a pair of duplicate dots with a distinctive recognition pattern, and reserving one pair per 4 1 4 array for ink spots for orientation, 7 plates each were spotted into 1 of the 6 fields of 1 filter, resulting in a total duplicate representation on 1 filter of 16,128 clones, each field containing 384 4 1 4 squares that gave 8 different duplicate patterns. The entire YAC library was thus represented on three 22 1 22 cm filters. Spotted filters were placed on top of (-ura) SD agar and allowed to grow for 1 –2 days at 307C such that colonies reached maximal size but no confluence occurred. Yeast cells were then spheroplasted by placing the filters on 2 pieces of blotting paper saturated with 45 ml SCE/DTT/zymolyase (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, 10 mM DTT, 5 unit/ml zymolyase 100T, ICN) in a Nunc bioassay plate and incubating overnight at 377C. Filters were then floated on denaturing buffer (0.5 M NaOH, 1.5 M NaCl) for 15 min, dried on blotting paper for 10 min, neutralized in 1 M Tris– HCl, 1.5 M NaCl, pH 7.4, for 5 min, placed in a 10-fold dilution of this neutralization buffer for an additional 5 min, treated for 45 min with proteinase K (50 mM EDTA, 100 mM NaCl, 50 mM Tris– HCl, 1% Sarkosyl, 0.25 mg/ml proteinase K, pH 8.0) at 377C, rinsed in 50 mM sodium phosphate buffer (pH 7.2), dried at room temperature, and UV cross-linked using a Stratagene apparatus. For Southern probing, YAC colony filters were prehybridized in
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Church hybridization buffer (0.5 M sodium phosphate, pH 7.2, 5% SDS, 1% BSA, 1 mM EDTA, 0.1 mg/ml yeast tRNA, 0.1 mg/ml herring sperm DNA) at 657C for 6 h. Probes were isotopically labeled using random primer labeling (T7 QuickPrimer Kit, Pharmacia) in the presence of [a-32P]dCTP, and competed with 1.5 mg/ml herring sperm DNA in 0.12 M Na2HPO 4 if the complexity of the probe required it. Filters were hybridized for 16 h at 657C, washed with 40 mM sodium phosphate buffer, 0.1% SDS at room temperature twice for 10 min each and once at 657C for 10 min. (d) Fluorescence in situ hybridization (FISH) analysis. To prepare degenerate, oligonucleotide-primered PCR (DOP-PCR) products as probes for FISH analysis, individual YAC plugs were prepared as outlined above, washed twice in 15 ml double-distilled H2O at room temperature, and left overnight with gentle agitation. For pre-DOPPCR a 3.5-ml aliquot from a melted YAC plug was amplified in a total volume of 5 ml containing 250 mM each dNTP, 10 pmol DOP primer (Telenius et al., 1992), 26 mM Tris– HCl, pH 9.5, 6.5 mM MgCl2 , and 2 units Thermo Sequenase DNA polymerase (Amersham, Life Science) on a Perkin– Elmer 9600 thermal cycler using the following protocol: 757C for 5 min, followed by 8 cycles of denaturation at 947C for 1 min, annealing at 307C for 1 min, and elongation at 747C for 2 min. The first reamplification was carried in a 50-ml reaction volume containing 5 ml of the pre-DOP reaction product, 50 mM KCl, 10 mM Tris – HCl, pH 9.0, 0.1% Triton X-100, 250 mM each dNTP, 10 pmol DOP primers, 2.5 mM MgCl2 , and 2 units Taq DNA polymerase (Promega). The reaction mix was denatured at 947C for 1 min and taken through 35 cycles of denaturation at 947C for 1 min, annealing at 407C for 1min, and elongation at 747C for 4 min, followed by a terminal elongation of 10 min at 727C. Finally, a second reamplification was carried out on 1 ml of the product from the first reamplification, using exactly the same conditions as before. Probes were labeled by nick translation to generate fragments between 100 and 500 bp in length. Four micrograms of DOP-PCR product was used for a 200-ml reaction mix containing 50 mM Tris– HCl, pH 8.0, 5 mM MgCl2 , 10 mg BSA, 0.01 mM b-mecaptoethanol, 50 mM dATP, dCTP, dGTP, 12.5 mM dUTP, 37.5 mM biotin-16– dUTP or DIG-11 –dUTP (Boehringer Mannheim), 0.05 units DNase I (Boehringer Mannheim), and 25 units DNA –Polymerase I (Boehringer Mannheim). This reaction was allowed to proceed at 167C for 2 h and was stopped by adding 2.5 ml 0.5 M EDTA (pH 8.0), 1.5 ml 10% SDS and by incubating at 687C for 10 min. Probes were purified by passage over Sephadex G-50 columns. Labeled probes are stable at 0207C. Metaphase chromosome slides were prepared from rat cell line cultures (TGR-1, Prouty et al., 1993) or freshly harvested rat spleen cells. Cells were pretreated with RNase A (100 mg/ml) in 21 SSC for 30 min at 377C and with pepsin (0.1 mg/ml) in 0.01 M HCl at 377C for 10 min, rinsed with PBS, and fixed for 10 min at room temperature in a PBS solution containing 1% formaldehyde and subsequent dehydration in a series of ethanol dilutions (70, 80, 90, and 100%). Pretreated slides were denatured at 907C for 1 min in the presence of a denaturation solution (21 SSC, 70% deionized formamide) on the surface of the slide, followed immediately by dehydration in a cold ethanol-dilution series. Four hundred nanograms of labeled probe was coprecipitated with 5 mg rat Cot-1 or 50–100 mg sheared rat genomic DNA as a competitor, dissolved in 30 ml hybridization solution (10% dextran sulfate, 50% deionized formamide), denatured at 807C for 10 min, and competed at 377C for 30 min. Precompeted probe was placed on the denatured slide, covered with a coverslip, sealed with rubber cement, and incubated at 377C in a moist chamber overnight. Slides were washed in 50% formamide, 21 SSC at 427C three times for 5 min each, washed in 0.11 SSC at 607C for 5 min, and blocked with 41 SSC, 3% BSA, 0.1% Tween at 377C for 30 min. Hybridization of biotinylated probes was detected with fluorescein isothiocyanate (FITC)-conjugated avidin. Digoxigenin-labeled probes were immunolocalized by means of rhodamine-labeled anti-digoxigenin antibodies. Chromosomes and cell nuclei were counterstained with 1 mg/ml DAPI in 21 SSC for 1 –5 min. The slides were mounted in 90% glycerol, 0.1 M Tris –HCl, pH 8.0, and 2.3% DABCO (1,4diazobicyclo-2,2,2-octane). Images were taken with a Zeiss epifluore
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scence microscope equipped with a cooled CCD camera (Photometrics PM50). Source images were captured separately with filter sets for FITC, rhodamine, and DAPI, saved as grayscale data files using the CCD Image capture software, and reproduced for analysis as enlarged hard copies using Oncor Image and Adobe Photoshop.
RESULTS AND DISCUSSION
Using the above-described set of methodologies we successfully constructed and characterized a large genomic YAC library, the first of its kind, for the rat. The techniques and methods employed, such as double size selection, were carefully chosen to result in a library with relatively large inserts, and a large number of transfectants were processed to reach a high degree of redundancy. We used a female stroke-prone, spontaneously hypertensive rat as source for DNA to arrive at an equitable representation of X-chromosomal DNA and because this laboratory has used this strain in the past for a large number of mapping studies directed at complex cardiovascular traits (Hilbert et al., 1991; Kreutz et al., 1995; Rubattu et al., 1996). The recent establishment of congenic lines for this strain (Kreutz et al., 1995) provided an added incentive to use representational difference analysis approaches (Lisitsyn et al., 1994). Since no embryonic stem cell system for gene targeting has yet been established in the rat, there was no particular predilection for any strain. The strain used is fully inbred, as assessed by typing with almost 500 polymorphic rat microsatellite markers. We used the vector pCGS966 (Smith et al., 1990) in preference to others as it permits copy number amplification of YACs (Smith, 1994). Several other features of this vector, such as the presence of two mirror-image restriction sites flanking an E. coli ori gene on both arms abutting the cloning site, as well as the presence of T3 and T7 promotors, and of a neo gene, make this a very versatile system. The first feature mentioned allows easy rescue of insert ends by restriction digest, circularizaton, and transformation of the resulting plasmids into a prokaryotic system, or by inverse PCR from the circularized product, while the neo gene permits direct transfection of YACs into eukaryotic systems. The T3 and T7 sites are useful for direct generation of riboprobes. The EcoRI cloning site of pCGS966 was used for the construction of the library, employing a partial digestion strategy for genomic DNA by competing the restriction endonuclease, EcoRI, against the corresponding EcoRI methylase, followed by two rounds of PFGE size selection for DNA fragments greater than 500 kb and removal of vector DNA. Conventional yeast spheroplasting was used for transformation, with transformation efficiencies showing a wide variation, from 5 to 10 3 transformants/mg DNA. The majority of the clones in the library were obtained from three different transformations. Transformants were plated on -ura plates, with most
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successful transformants visible after 2 days of growth. Plates were stored at 47C and individual YAC clones were lifted manually one by one into the 96-well microtiter plates containing (-ura, -trp) SD medium to select for both vector arms as an additional, more stringent selection step (the absence of a sup4 gene in pCGS966 does not permit adenenin-dependent pink/white selection in this vector; Smith, 1994). Double-nutrient-deficient selection resulted in the exclusion of about 5– 15% of colonies derived from single-selection medium. Colonies normally grew to saturation within 3 days and were processed for long-term, deep-freezer storage by suspending them in 40% SD, 40% YPD, 20% glycerol. We lifted 51,360 clones arrayed in 535 96-well microtiter plates; subtracting the empty wells, the library represents approximately 45,000 true insert-containing clones. In addition to arraying the library in this fashion, we processed the library to provide access to two screening methods by assembling PCR pools (67 secondary pools, representing a total of 1875 primary pools for unequivocal two-step address determination) and by producing high-density spotted filters for probe hybridization. The average insert size of this rat YAC library, determined from DNA preparation, PFGE, and Southern blotting against total genomic rat DNA on 166 randomly chosen clones was 736 kb (after subtraction of the vector arm size). This represents a conservative estimate as YAC clones that showed two or more bands were excluded from this analysis (these phenomena may represent cotransformation of several YACs into one colony, cross-contamination during picking and storage stages, or unstable YACs). Twelve percent of examined clones appeared to carry no insert; they might represent either the result of imperfect dephosphorylation of vector arms and carry-over during the second PFGE size-selection step or could be small-insert YAC clones indistinguishable under the PFGE conditions used. Figure 1 illustrates the distribution of YAC insert size, which, as expected under the constraints of double-size selection, is skewed toward the selected size cut-off. Thus, the majority of clones (88%) range from 200 to 1100 kb, with the mode in the range of 500–700 kb. The long tail from 1100 to 2000 kb and a small second mode of YACs ú2000 kb counterbalance the presence of small-insert clones still present after two rounds of size selection for a compression zone of ú500 kb (Birren and Lai, 1993) to account for the mean size of 736 kb. Due presumably to the inclusion of polyamines (Larin et al., 1991) and salt in several steps of our protocol (see Materials and Methods) to protect DNA from degradation, and due to the extremely careful handling throughout the entire library construction process, a relatively large proportion (20%) of megabase YAC clones are represented in our library. Taking both empty wells and empty clones into account, we arrive at an estimated size of our library of approximately 40,000 viable, large-insert clones. Given
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FIG. 1. Histogram showing size distribution for rat YAC inserts from 166 randomly chosen clones. Clones with more than one band and without inserts (about 12%) are not included.
the demonstrated average insert size and an assumed haploid mammalian genome size of 3 1 109 bp for the rat, we calculate that our YAC library contains 9.8 genome equivalents, thus providing 99.99% probability of containing a given DNA sequence (Sambrook et al., 1989). Since the DNA source was a female spleen, this coverage should apply to all 20 autosomes and the X chromosome. We validated our estimates regarding representation and complexity of the library by screening the library with genetic probes and microsatellite markers, using both Southern hybridization on high-density spotted filters and PCR pool amplification. Highly efficient screening methods are required to locate positive clones in libraries of the size of the one described here. Using highly processive and accurate robotic tools we were able to compress the entire library on three 22 1 22 cm filters, providing double representation (for specificity) as well as orientation markers. Among the advantages of high-density filter hybridization are the immediate localization of positive clones after only one round of probing, as well as the option to use several probes, such as from a number of cosmids, simultaneously and locate all YACs mapping to a particular contig at once. We tested mixed probes from the genes encoding kallikrein and muscle myosin light chain 2 and found a total of 23 positive clones on the 3 filters, consistent with our estimate of 10-fold genome coverage (Fig. 2). PCR-based screening strategies have the advantage of convenience, lack of need to label probes (radioisotopically or otherwise), and an ever-growing abundance of SSLP and STS markers to be used as probes. The theoretical disadvantage or limitation of the PCR-pooling approach relates to the diminishing relative representation of any given clone or sequence in a complex
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mixture of clones. The 3-dimensional pooling scheme we used results in superpools that contain a maximum of 768 clones each (assuming no empty wells), or about 1 6 of a genome-equivalent. Contrasting the genome size of AB1380 of about 14 Mb to the average insert size of 736 kb, any given YAC insert sequence would be present only once in a background of 11,316 Mb of total DNA (10,752 Mb of yeast chromosomes, 0.564 Mb of YACs), resulting, effectively, in a mass ratio about four times less advantageous than the mammalian genome. This raises concern about the proper representation of, and ability to find, sequences of interest. Using 48 different rat microsatellite primer pairs, we were gratified to find an average of 9.3 positive, specific signals per primer pair, which again is in keeping with our calculated size and representation of the library (Fig. 3). The well-appreciated avidity for homologous recombination in the yeast genome, along with coligation or cotransformation events during the library construction process, makes chimeric clones and clones with deletions or other rearrangements a frequently observed phenomenon in all YAC libraries. Thus, in most libraries constructed to date about one-third to onehalf of all clones are chimeric. While recombinationdeficient strains of Saccharomyces cerevisiae have been available for some time, their transformation efficiency remains poor, and no large library has been constructed in any of these strains so far; therefore, we used the conventional AB1380 strain despite its disadvantages. To assess the proportion of chimeric clones present in our library we performed FISH analysis on 39 randomly chosen YACs (from among those that yielded only a single band on Southern analysis with total rat DNA). Of those, 8 showed clear evidence of chimerism with pairs of signals on different homologous chromo-
FIG. 2. Library screening by hybridization. Two probes were pooled and used to screen the entire library (three 22 1 22 cm filters, one of which is shown). Positive clones could be identified by the characteristic double signal within one 4 1 4 dot array.
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FIG. 3. PCR-pool screening of the rat YAC library for clones containing the microsatellite marker D1Mgh7. (Top) Agarose gel analysis of PCR amplification of 67 secondary pools. Two negative controls (host yeast genomic DNA in the left-most lane of the upper row, and no DNA in the right-most lane, upper row) and two positive controls (total rat genomic DNA in the second lane from the left in both upper and lower row of wells). Nine positive secondary pools for this marker are found. (Bottom) Agarose gel analysis of 28 primary DNA pools representing one of the positive secondary pools. Left-most and right-most lanes contain a 100-bp molecular weight marker, lanes 2 –9 (from the left) represent the 8 plate pools, lanes 10–17 represent the 8 row-pools, and lane 18 –29 represent the 12 column pools. Lane 30 contains a positive control (total rat genomic DNA) and lane 31, the respective secondary pool represented in this analysis. From the amplification pattern the address of the positive clone is plate 1, row 5, and column 5.
somes. Four YACs produced multiple (more than 4) hybridization signals at chromosomal sites known to contain repeat DNA sequences and are thus likely to carry satellite DNA or rDNA sequences, whereas the remaining 27 YACs produced only a single pair of signals on homologous chromosomes (Fig. 4). These results place our library right within the range of about 20–30% chimerism that would be predicted by past experience. DOP-PCR has been demonstrated to produce signals more uniformly along the chromosomes than interspersed repetitive sequence PCR (IRS-PCR); thus the likelihood that we missed chimerism due to biased amplification is low and we believe our estimate to be a realistic one. Genomic libraries are essential tools for the analysis of complex genomes and the discovery of novel genes,
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particularly genes localized by positional cloning. Different cloning capacity, host and vector systems, degree of manipulation possible, and ease or difficulty of construction determine the special features of various cloning systems, usually making different cloning systems advisable for different stages of genome analysis. Among large-insert genomic cloning systems, YACs have been widely used during the initial stage of positional cloning when the resolving power of linkage analysis usually defines only a relatively large window and have proven to be superior tools in the assembly of far-spanning contigs and the construction of physical maps. Since the original description of the technique (Burke et al., 1987), a large number of libraries have been constructed in different species, including human (Albertsen et al., 1990; Anand et al., 1990; Brownstein et al., 1989; Larin et al., 1991; Traver et al., 1989), mouse (Larin et al., 1991; Rossi et al., 1992), Caenorhabditis elegans (Coulson et al., 1988), Drosophila melanogaster (Garza et al., 1989), commercially important agricultural animals such as cattle (Libert et al., 1993) and pig, (Leeb et al., 1995), as well as in plants (tomato, maize, Arabidopsis). A large number of genes owe their detection to the availability of these libraries, despite their drawbacks as noted above, such as low cloning efficiency, chimerism, and difficulty of handling. The ability of YACs to carry, by a wide margin, the largest genomic inserts makes this system still an essential one, particularly for as yet poorly characterized genomes such as the rat. The value of YACs is of course further augmented by the concomitant availability of other, complementary libraries in different cloning systems, such as BACs (Shizuya et al., 1992)
FIG. 4. Fluorescence in situ hybridization of rat metaphase chromosomes for a randomly chosen YAC clone.
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and PACs (Ioannou et al., 1994), which are based on the E. coli F factor and afford high cloning efficiency, a low degree of chimerism, and relative ease of handling (Shizuya et al., 1992; Woo et al., 1994; Cai et al., 1995; Wang et al., 1994; Kim et al., 1996). Generating such libraries and integrating them with the currently created YAC library will be the next important task we face in creating a comprehensive set of genomic resources for the laboratory rat. ACKNOWLEDGMENTS We thank Kevin Benjamin, Sridharam Raghavan, Humphrey Wattanga, and Carl Mark for their technical assistance in arraying the YAC library. This work was supported by a Research Career Development Award (K04-HL03138-01) from the National Heart, Lung, and Blood Institute to K.L.; by Grant GA175/8-1 from the German Research Council (DFG); by Grant 01KW9607 from the German Ministry for Education, Science, Research and Technology (BMBF), and by a grant from the EC Biotech Program. Resource availability. High-density spotted filters are available through the Resource Center/Primary Database (RZPD) of the German Human Genome Project (Website address: http://rzpd.rzberlin.mpg.de/). PCR pools and screening are available through Research Genetics Inc. (AL, USA; Website address: http://www.resgen.com/).
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Traver, C. N., Klapholz, S., Hyman, R. W., and Davis, R. W. (1989). Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences. Proc. Natl. Acad. Sci. USA 86: 5898 –5902. Wang, M., Chen, X.-N., Shouse, S., Manson, J., Wu, Q, Li, R., Wrestler, Noya, D., Sun, Z.-G., Korenberg, J., and Lai, E. (1994). Construction and characterization of a human chromosome 2-specific BAC library. Genomics 24: 527– 534. Woo, S.-S., Jiang, J., Gill, B. S., Paterson, A. H., and Wing, R. A. (1994). Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. Nucleic Acids Res. 22: 4922 –4931. Yamada, J., Kuramoto, T., and Serikawa, T. (1994). A rat genetic linkage map and comparative maps for mouse or human homologous rat genes. Mamm. Genome 5: 63–83.
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ARTICLE NO.
Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat, Rattus norvegicus LI CAI,* LEONARD C. SCHALKWYK,† ANDREINA SCHOEBERLEIN-STEHLI,‡ ROBERT Y. L. ZEE,* AVRIAL SMITH,† THOMAS HAAF,† MICHEL GEORGES,‡ HANS LEHRACH,† AND KLAUS LINDPAINTNER*,§,1 *Cardiovascular Division, Brigham and Women’s Hospital, Department of Cardiology, Children’s Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; †Max Planck Institute for Molecular Genetics, Berlin– Dahlem, Germany; ‡Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Lie´ge, Lie´ge, Belgium; and §Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany Received August 23, 1996; accepted October 28, 1996
Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20– 30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. q 1997 Academic Press
INTRODUCTION
The laboratory rat, Rattus norvegicus, is one of the most widely studied experimental organisms in biomedical research. The rat’s size — large enough to allow complex physiological measurements, yet small enough 1 To whom correspondence should be addressed at Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis Street, Thorn 1103, Boston, MA 02115. Telephone: (617) 732-8173. Fax: (617) 2646830. E-mail: [email protected].
for economical breeding— and the availability of a large number of inbred strains have made it a favored animal in physiological research (Hedrich, 1990). More recently, the rat has been recognized as an important experimental system for genetic studies of multifactorial disease, such as hypertension, stroke, diabetes, and renal failure. These strains represent a useful adjuvant to the study of complex disease in humans, as they preserve the polygenic character of the disorder, but allow a reduction of complexity provided by the lack of genetic heterogeneity, by standardization of environmental influences, and by the imposition of programmed breeding schemes. Thus, the feasibility of mapping quantitative trait loci (QTL) in mammals was first demonstrated in cross-breeding experiments with inbred strains of rats (Hilbert et al., 1991; Jacob et al., 1991). However, the lack of genomic resources for the rat —as compared to human or mouse— has significantly impeded progress toward identification of traitrelevant genes. The availability of polymorphic markers — while continuously improving — is still scarce, and the map of the rat genome remains rather sketchy (Levan et al., 1991; Serikawa et al., 1992; Yamada et al., 1994; Jacob et al., 1995; Pravenec et al., 1996; http:// www.RATMAP). To utilize fully the emerging panels of more densely spaced polymorphic markers, as well as to aid in the development of targeted markers, a set of large-insert genomic libraries (yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), cosmids) is an essential requirement. Here, we report the establishment and characterization of the first YAC library for the rat. MATERIALS AND METHODS
1. Construction of a Rat YAC Library (a) Preparation of high-molecular-weight DNA. A fresh spleen was taken from a stroke-prone, spontaneously hypertensive rat (a
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0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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fully inbred strain commonly used in cardiovascular research) and cut in half. Cells were extruded by blunt manipulation into a petri dish containing 20 ml ice-cold phosphate-buffered saline (PBS: 1% NaCl, 0.025% KCl, 0.0143% Na2HPO4 , 0.0025% KH2PO4). Spleen cells were suspended in 50 ml PBS, and coarse tissue debris was allowed to settle by gravity for 5 min. The supernatant cell suspension was transferred to a new 50-ml Falcon tube and cells were pelleted by centrifugation at 600g for 10 min at 47C. The pellet was resuspended by gentle agitation in 50 ml cold PBS and counted using a hemocytometer. Nucleated cell number was estimated by multiplying the raw data by a factor of 0.6 as an empirically found adjustment to account for the presence of erythrocytes. Cells were re-pelleted by centrifugation as described and resuspended in PBS to yield a concentration of 4 1 107 cells/ml. This cell suspension was mixed with an equal volume of 1.5% low-melting-point agarose (dissolved in PBS) at 407C and cast as 0.75% agarose plugs into 100-ml molds (Bio-Rad). Plugs were equilibrated in ESP solution (0.5 M EDTA, 1% N-lauroylsarcosine sodium, 1 mg/ml proteinase K) and incubated at 507C for 48 h with gentle shaking. Plugs were subsequently washed against TE (10 mM Tris – HCl, 1 mM EDTA, pH 8.0) at 507C for 30 min, then against 1 mM PMSF in TE at 507C for 30 min, and finally against TE at room temperature. Plugs were stored in 0.5 M EDTA at 47C until use, when they were again equilibrated against 11 TE. (b) YAC vector preparation. The 19.5-kb vector, pCGS966 (19.5 kb, Smith et al., 1990), transformed into Escherichia coli strain XL1blue [recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac (F* proAB lacIq lacZDM15 Tn10], was used for construction of the library. Transformed cultures were grown overnight at 377C on LB agar plates containing 50 mg/ml ampicillin. Four single colonies were picked, and plasmid DNA was prepared and digested with HindIII for agarose-gel verification that no telomeric deletions had occurred during propagation in E. coli. Vector DNA was prepared using CsCl gradient centrifugation (Sambrook et al., 1989). For cloning, the vector was digested to completion with EcoRI and BamHI, as verified by agarose gel electrophoresis. DNA was dephosphorylated at 377C for 40 min using calf intestinal phosphatase (Boehringer Mannheim). Digestion products were purified using StrataClean Resin (Stratagene), precipitated, and dissolved in TE. Completeness of dephosphorylation was verified by the absence of a ligation product on agarose gel electrophoresis after overnight incubation of 200 ng of dephosphorylated DNA in the presence of DNA ligase, ATP, and Mg/ at 167C. Additional quality controls were carried out by transforming unrestricted vector, restricted vector, and restricted, phophatased, rekinased, and religated vector into E. coli. (c) Partial digestion of genomic DNA. Partially digested genomic DNA was produced by concomitant incubation with both the restriction endonuclease, EcoRI (NewEngland BioLab), and the corresponding EcoRI methylase (NEB). One agarose plug (containing about 12 mg genomic DNA) was equilibrated against 400 ml EcoRI digestion mix (80 mM S-adenosylmethionine, 2 mM MgCl2 , 100 mM NaCl, 50 mM Tris –HCl, pH 7.5, 1 mM DTT, 0.5 mg/ml BSA, 4 mM spermidine, and variable amounts of EcoRI/EcoRI methylase) on ice for 30 min. The partial digestion reaction was started by transferring the tubes to a 377C water bath for 4 h and was stopped by adding 30 ml 0.5 M EDTA (pH 8.0). The ratio of EcoRI to EcoRI methylase was adjusted (EcoRI/EcoRI methylase: 1/60– 1/70) to yield a majority of DNA fragments in the size range above 500 kb. (d) First size selection. The partially digested DNA was electrophoretically size-fractionated on a 1% low-melting agarose gel (FMC, Seaplaque GTG) using a CHEF apparatus (Bio-Rad). Megabase-size DNA was separated in 0.51 TBE buffer (11 TBE is 0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8.0) at 117C, 6 V/cm for 16 h, with a 25-s switch time, resulting in the concentration of all DNA fragments larger than 500 kb in the compression zone. The compression band was excised for further manipulation. (e) Ligation. The gel piece containing the compression band was dialyzed three times for 30 min each against ice-cold 11 ligation buffer (50 mM Tris –HCl, pH 7.5, 10 mM MgCl2 , 30 mM NaCl, 0.75 mM spermidine, 0.3 mM spermine). Vector DNA prepared as detailed
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above was then added in approximately 20 molar excess. The sample was heated to 687C for 10 min, mixed by gentle inversion, and incubated at 377C for 1 h to allow for uniform distribution of vector and genomic DNA. The ligation reaction was started by adding DTT (1 mM ), ATP (10 mM ), and 200 units of ligase per milliliter (NEB), followed by gentle inversion of the tube, and the reaction was completed by overnight incubation at room temperature. (f) Second size selection. The solidified low-melting agarose gel block containing the ligation product was cut into 2-mm-thick slices that were equilibrated against ice-cold 0.51 TBE with two changes of buffer, for 30 min each. Slices were then loaded on a 1% lowmelting agarose gel and size selection was carried out analogously to the first size selection. The compression band was excised equilibrated against ice-cold TENP buffer (10 mM Tris– HCl, pH 7.5, 1 mM EDTA, 30 mM NaCl, 0.75 mM spermidine, 0.3 mM spermine) three times, for 30 min each. To prepare the DNA sample for transformation, the gel slices were melted at 687C for 10 min, and equilibrated at 407C for 10 min, and 5 units of beta-agarase I (NEB) was added for each 1 ml of gel volume, with a subsequent incubation at 407C for 2 h. (g) Transformation. We modified the protocol communicated by Burgers and Percival (1987), using zymolyase (20T, ICN), at 8– 22 units per 50 ml of yeast cells grown to an OD600 of 4.0 instead of lyticase. Likewise, SPEM (1 M sorbitol, 1.6 mM NaH2PO4, 8.4 mM Na2HPO4, 5 mM EDTA. 0.2% b-mercaptoethanol) was used instead of SCEM (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, 30 mM b-mercaptoethanol). Transformants were selected on uracildeficient medium and were visible after 2 –3 days. (h) Storage of the YAC library. Single colonies were transferred manually with sterile toothpicks into 96-well microtiter plates containing 180 ml SD medium [2% glucose, 0.072% CSM -trp -ura, (BIO101), 0.67% yeast nitrogen base without amino acids, (DIFCO)] and were grown for 3 days at 307C. As a consequence of the more stringent double-selective medium (-trp, -ura), about 5– 15% of wells failed to show growth. These empty wells were recorded. After mixing in an additional 180 ml storage medium (YPD with 40% glycerol; YPD is 1% yeast extract, 2% bactopeptone, 2% glucose) using a 12channel pipette, three master-aliquots of 100 ml each were prepared and frozen at 0807C. A single replica plate made using a 96-pin replicator was likewise allowed to grow and was split into three aliquots containing 100 ml each.
2. Characterization of the YAC Library We used a combination of several approaches to determine qualitative and quantitative characteristics of the library, including measurements of average insert size, screening of the library by PCR using pools and by Southern hybridization using high-density spotted filters, and fluorescence in situ hybridizaton on karyograms to determine the fraction of chimeric clones. (a) Average insert size determination. Randomly selected YAC clones were grown in 5 ml SD medium for 3 days at 307C in a shaking incubator. Two 100-ml agarose plugs were made from the culture as previously described (Sambrook et al., 1989). Half of a plug was placed into the sample slot of a pulsed-field gel and run for 18 h at 6 V/cm, 117C, and with a switch time of 60 s. After depurination with 0.2 M HCl for 30 min the gel was blotted against a nylon membrane (Hybond-N/, Amersham). Total rat genomic DNA, sheared and radiolabeled with [a-32 P]dCTP using Klenow polymerase in the presence of random hexamer primers, was used as a probe to detect the inserts. A standard yeast chromosome marker was included to determine the insert size. (b) Preparation of PCR screening pools. A 3-dimensional pooling strategy was used to prepare DNA pools for PCR screening. Three copies of the YAC library (replicas from one of the six master copies) were grown in 96-well microtiter plates containing 200 ml SD medium per well, at 307C for 3 days. Eight 96-well microtiter plates were grouped as a block and colonies were pooled using an 8- and a 12channel manifold vacuum device (Sigma) to combine wells from one
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RAT YAC LIBRARY row (x/y plane, yielding 8 row pools) or one column (y/z plane, yielding 12 column pools) from all 8 plates, or wells from one entire plate (x/ z plane, 8 plate pools), providing a total of 28 primary pools from each block for unequivocal addressing of a single well among all 768 contained within the block. DNA was extracted using a previously described method (Sambrook et al., 1989). In brief, pooled cell suspensions (24 ml from plate and row pools and 16 ml from column pools) were centrifuged at 600g for 5 min to pellet yeast cells. Cell pellets were resuspended in 5 ml 50 mM EDTA (pH 8.0) and pelleted as before. The supernatant was discarded, and the cell pellet was dried at room temperature for 10 min and then resuspended in 200 ml lyticase solution (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, pH 8.0, 5% b-mecaptoethanol, and 80 units of lyticase). Following a 20-min incubation at room temperature, 200 ml of 2% lowmelting-point agarose in 125 mM EDTA, pH 8.0, was added, mixed with the cell lysate by gentle agitation, and allowed to solidify at room temperature for 20 min. The resulting agarose plugs were suspended in 2.5 ml 50 mM EDTA, pH 8.0, containing 5% b-mercaptoethanol, and incubated at 307C overnight. After removal of this solution, the agarose plugs were incubated in 2.5 ml ESP solution (450 mM EDTA, pH 8.0, 1% SDS, 1 mg/ml proteinase K) at 507C for 36 h and then washed once with TE, containing 1 mM PMSF, and twice with plain TE. The plugs were melted at 407C after adding 600 ml TE and finally incubated with agarase (Sigma). Secondary (‘‘super’’) pools were generated by pooling equal portions of all 28 primary pool DNA samples from each block of 8 plates. A total of 67 secondary pools were thus generated from 1875 primary pools, representing 535 96-well YAC plates. All primary and secondary pools were stored in deep-well 96-titer plates (Beckman) at 0807C. Initial quality assessment of the YAC pools was carried out by interrepetitive sequences (IRS)-PCR using primers derived from the rat ID element, revealing a characteristic smear from the secondary DNA pools and several distinct bands from the primary DNA pools. Subsequent screening with locus-specific primer pairs was conducted according to a two-stage approach by first amplifying from secondary pools and then determining the specific clone address by subsequent PCR using the respective set of 28 primary pools. (c) Preparation of high-density grid-spotted filters. To allow the use of high-throughput robotic processing, we first generated a copy of the library in 384-well format by replicating four 96-well YAC plates each into one 384-well microtiter plate (Genetix) filled with (ura) SD medium using a 96-pin replicator (Genetix). This spotting copy was allowed to grow for 3 days at 307C. A 4 1 4 duplicate pattern was used for generation of the high-density filters on 22 1 22 cm nylon membranes (Hybond-N/, Amersham). Each filter provided 6 fields corresponding in size to the footprint of one 384-well plate; the area of each single well was represented on the filter by a 4 1 4 array of 16 dots. By spotting each well as a pair of duplicate dots with a distinctive recognition pattern, and reserving one pair per 4 1 4 array for ink spots for orientation, 7 plates each were spotted into 1 of the 6 fields of 1 filter, resulting in a total duplicate representation on 1 filter of 16,128 clones, each field containing 384 4 1 4 squares that gave 8 different duplicate patterns. The entire YAC library was thus represented on three 22 1 22 cm filters. Spotted filters were placed on top of (-ura) SD agar and allowed to grow for 1 –2 days at 307C such that colonies reached maximal size but no confluence occurred. Yeast cells were then spheroplasted by placing the filters on 2 pieces of blotting paper saturated with 45 ml SCE/DTT/zymolyase (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10 mM EDTA, 10 mM DTT, 5 unit/ml zymolyase 100T, ICN) in a Nunc bioassay plate and incubating overnight at 377C. Filters were then floated on denaturing buffer (0.5 M NaOH, 1.5 M NaCl) for 15 min, dried on blotting paper for 10 min, neutralized in 1 M Tris– HCl, 1.5 M NaCl, pH 7.4, for 5 min, placed in a 10-fold dilution of this neutralization buffer for an additional 5 min, treated for 45 min with proteinase K (50 mM EDTA, 100 mM NaCl, 50 mM Tris– HCl, 1% Sarkosyl, 0.25 mg/ml proteinase K, pH 8.0) at 377C, rinsed in 50 mM sodium phosphate buffer (pH 7.2), dried at room temperature, and UV cross-linked using a Stratagene apparatus. For Southern probing, YAC colony filters were prehybridized in
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Church hybridization buffer (0.5 M sodium phosphate, pH 7.2, 5% SDS, 1% BSA, 1 mM EDTA, 0.1 mg/ml yeast tRNA, 0.1 mg/ml herring sperm DNA) at 657C for 6 h. Probes were isotopically labeled using random primer labeling (T7 QuickPrimer Kit, Pharmacia) in the presence of [a-32P]dCTP, and competed with 1.5 mg/ml herring sperm DNA in 0.12 M Na2HPO 4 if the complexity of the probe required it. Filters were hybridized for 16 h at 657C, washed with 40 mM sodium phosphate buffer, 0.1% SDS at room temperature twice for 10 min each and once at 657C for 10 min. (d) Fluorescence in situ hybridization (FISH) analysis. To prepare degenerate, oligonucleotide-primered PCR (DOP-PCR) products as probes for FISH analysis, individual YAC plugs were prepared as outlined above, washed twice in 15 ml double-distilled H2O at room temperature, and left overnight with gentle agitation. For pre-DOPPCR a 3.5-ml aliquot from a melted YAC plug was amplified in a total volume of 5 ml containing 250 mM each dNTP, 10 pmol DOP primer (Telenius et al., 1992), 26 mM Tris– HCl, pH 9.5, 6.5 mM MgCl2 , and 2 units Thermo Sequenase DNA polymerase (Amersham, Life Science) on a Perkin– Elmer 9600 thermal cycler using the following protocol: 757C for 5 min, followed by 8 cycles of denaturation at 947C for 1 min, annealing at 307C for 1 min, and elongation at 747C for 2 min. The first reamplification was carried in a 50-ml reaction volume containing 5 ml of the pre-DOP reaction product, 50 mM KCl, 10 mM Tris – HCl, pH 9.0, 0.1% Triton X-100, 250 mM each dNTP, 10 pmol DOP primers, 2.5 mM MgCl2 , and 2 units Taq DNA polymerase (Promega). The reaction mix was denatured at 947C for 1 min and taken through 35 cycles of denaturation at 947C for 1 min, annealing at 407C for 1min, and elongation at 747C for 4 min, followed by a terminal elongation of 10 min at 727C. Finally, a second reamplification was carried out on 1 ml of the product from the first reamplification, using exactly the same conditions as before. Probes were labeled by nick translation to generate fragments between 100 and 500 bp in length. Four micrograms of DOP-PCR product was used for a 200-ml reaction mix containing 50 mM Tris– HCl, pH 8.0, 5 mM MgCl2 , 10 mg BSA, 0.01 mM b-mecaptoethanol, 50 mM dATP, dCTP, dGTP, 12.5 mM dUTP, 37.5 mM biotin-16– dUTP or DIG-11 –dUTP (Boehringer Mannheim), 0.05 units DNase I (Boehringer Mannheim), and 25 units DNA –Polymerase I (Boehringer Mannheim). This reaction was allowed to proceed at 167C for 2 h and was stopped by adding 2.5 ml 0.5 M EDTA (pH 8.0), 1.5 ml 10% SDS and by incubating at 687C for 10 min. Probes were purified by passage over Sephadex G-50 columns. Labeled probes are stable at 0207C. Metaphase chromosome slides were prepared from rat cell line cultures (TGR-1, Prouty et al., 1993) or freshly harvested rat spleen cells. Cells were pretreated with RNase A (100 mg/ml) in 21 SSC for 30 min at 377C and with pepsin (0.1 mg/ml) in 0.01 M HCl at 377C for 10 min, rinsed with PBS, and fixed for 10 min at room temperature in a PBS solution containing 1% formaldehyde and subsequent dehydration in a series of ethanol dilutions (70, 80, 90, and 100%). Pretreated slides were denatured at 907C for 1 min in the presence of a denaturation solution (21 SSC, 70% deionized formamide) on the surface of the slide, followed immediately by dehydration in a cold ethanol-dilution series. Four hundred nanograms of labeled probe was coprecipitated with 5 mg rat Cot-1 or 50–100 mg sheared rat genomic DNA as a competitor, dissolved in 30 ml hybridization solution (10% dextran sulfate, 50% deionized formamide), denatured at 807C for 10 min, and competed at 377C for 30 min. Precompeted probe was placed on the denatured slide, covered with a coverslip, sealed with rubber cement, and incubated at 377C in a moist chamber overnight. Slides were washed in 50% formamide, 21 SSC at 427C three times for 5 min each, washed in 0.11 SSC at 607C for 5 min, and blocked with 41 SSC, 3% BSA, 0.1% Tween at 377C for 30 min. Hybridization of biotinylated probes was detected with fluorescein isothiocyanate (FITC)-conjugated avidin. Digoxigenin-labeled probes were immunolocalized by means of rhodamine-labeled anti-digoxigenin antibodies. Chromosomes and cell nuclei were counterstained with 1 mg/ml DAPI in 21 SSC for 1 –5 min. The slides were mounted in 90% glycerol, 0.1 M Tris –HCl, pH 8.0, and 2.3% DABCO (1,4diazobicyclo-2,2,2-octane). Images were taken with a Zeiss epifluore
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scence microscope equipped with a cooled CCD camera (Photometrics PM50). Source images were captured separately with filter sets for FITC, rhodamine, and DAPI, saved as grayscale data files using the CCD Image capture software, and reproduced for analysis as enlarged hard copies using Oncor Image and Adobe Photoshop.
RESULTS AND DISCUSSION
Using the above-described set of methodologies we successfully constructed and characterized a large genomic YAC library, the first of its kind, for the rat. The techniques and methods employed, such as double size selection, were carefully chosen to result in a library with relatively large inserts, and a large number of transfectants were processed to reach a high degree of redundancy. We used a female stroke-prone, spontaneously hypertensive rat as source for DNA to arrive at an equitable representation of X-chromosomal DNA and because this laboratory has used this strain in the past for a large number of mapping studies directed at complex cardiovascular traits (Hilbert et al., 1991; Kreutz et al., 1995; Rubattu et al., 1996). The recent establishment of congenic lines for this strain (Kreutz et al., 1995) provided an added incentive to use representational difference analysis approaches (Lisitsyn et al., 1994). Since no embryonic stem cell system for gene targeting has yet been established in the rat, there was no particular predilection for any strain. The strain used is fully inbred, as assessed by typing with almost 500 polymorphic rat microsatellite markers. We used the vector pCGS966 (Smith et al., 1990) in preference to others as it permits copy number amplification of YACs (Smith, 1994). Several other features of this vector, such as the presence of two mirror-image restriction sites flanking an E. coli ori gene on both arms abutting the cloning site, as well as the presence of T3 and T7 promotors, and of a neo gene, make this a very versatile system. The first feature mentioned allows easy rescue of insert ends by restriction digest, circularizaton, and transformation of the resulting plasmids into a prokaryotic system, or by inverse PCR from the circularized product, while the neo gene permits direct transfection of YACs into eukaryotic systems. The T3 and T7 sites are useful for direct generation of riboprobes. The EcoRI cloning site of pCGS966 was used for the construction of the library, employing a partial digestion strategy for genomic DNA by competing the restriction endonuclease, EcoRI, against the corresponding EcoRI methylase, followed by two rounds of PFGE size selection for DNA fragments greater than 500 kb and removal of vector DNA. Conventional yeast spheroplasting was used for transformation, with transformation efficiencies showing a wide variation, from 5 to 10 3 transformants/mg DNA. The majority of the clones in the library were obtained from three different transformations. Transformants were plated on -ura plates, with most
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successful transformants visible after 2 days of growth. Plates were stored at 47C and individual YAC clones were lifted manually one by one into the 96-well microtiter plates containing (-ura, -trp) SD medium to select for both vector arms as an additional, more stringent selection step (the absence of a sup4 gene in pCGS966 does not permit adenenin-dependent pink/white selection in this vector; Smith, 1994). Double-nutrient-deficient selection resulted in the exclusion of about 5– 15% of colonies derived from single-selection medium. Colonies normally grew to saturation within 3 days and were processed for long-term, deep-freezer storage by suspending them in 40% SD, 40% YPD, 20% glycerol. We lifted 51,360 clones arrayed in 535 96-well microtiter plates; subtracting the empty wells, the library represents approximately 45,000 true insert-containing clones. In addition to arraying the library in this fashion, we processed the library to provide access to two screening methods by assembling PCR pools (67 secondary pools, representing a total of 1875 primary pools for unequivocal two-step address determination) and by producing high-density spotted filters for probe hybridization. The average insert size of this rat YAC library, determined from DNA preparation, PFGE, and Southern blotting against total genomic rat DNA on 166 randomly chosen clones was 736 kb (after subtraction of the vector arm size). This represents a conservative estimate as YAC clones that showed two or more bands were excluded from this analysis (these phenomena may represent cotransformation of several YACs into one colony, cross-contamination during picking and storage stages, or unstable YACs). Twelve percent of examined clones appeared to carry no insert; they might represent either the result of imperfect dephosphorylation of vector arms and carry-over during the second PFGE size-selection step or could be small-insert YAC clones indistinguishable under the PFGE conditions used. Figure 1 illustrates the distribution of YAC insert size, which, as expected under the constraints of double-size selection, is skewed toward the selected size cut-off. Thus, the majority of clones (88%) range from 200 to 1100 kb, with the mode in the range of 500–700 kb. The long tail from 1100 to 2000 kb and a small second mode of YACs ú2000 kb counterbalance the presence of small-insert clones still present after two rounds of size selection for a compression zone of ú500 kb (Birren and Lai, 1993) to account for the mean size of 736 kb. Due presumably to the inclusion of polyamines (Larin et al., 1991) and salt in several steps of our protocol (see Materials and Methods) to protect DNA from degradation, and due to the extremely careful handling throughout the entire library construction process, a relatively large proportion (20%) of megabase YAC clones are represented in our library. Taking both empty wells and empty clones into account, we arrive at an estimated size of our library of approximately 40,000 viable, large-insert clones. Given
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FIG. 1. Histogram showing size distribution for rat YAC inserts from 166 randomly chosen clones. Clones with more than one band and without inserts (about 12%) are not included.
the demonstrated average insert size and an assumed haploid mammalian genome size of 3 1 109 bp for the rat, we calculate that our YAC library contains 9.8 genome equivalents, thus providing 99.99% probability of containing a given DNA sequence (Sambrook et al., 1989). Since the DNA source was a female spleen, this coverage should apply to all 20 autosomes and the X chromosome. We validated our estimates regarding representation and complexity of the library by screening the library with genetic probes and microsatellite markers, using both Southern hybridization on high-density spotted filters and PCR pool amplification. Highly efficient screening methods are required to locate positive clones in libraries of the size of the one described here. Using highly processive and accurate robotic tools we were able to compress the entire library on three 22 1 22 cm filters, providing double representation (for specificity) as well as orientation markers. Among the advantages of high-density filter hybridization are the immediate localization of positive clones after only one round of probing, as well as the option to use several probes, such as from a number of cosmids, simultaneously and locate all YACs mapping to a particular contig at once. We tested mixed probes from the genes encoding kallikrein and muscle myosin light chain 2 and found a total of 23 positive clones on the 3 filters, consistent with our estimate of 10-fold genome coverage (Fig. 2). PCR-based screening strategies have the advantage of convenience, lack of need to label probes (radioisotopically or otherwise), and an ever-growing abundance of SSLP and STS markers to be used as probes. The theoretical disadvantage or limitation of the PCR-pooling approach relates to the diminishing relative representation of any given clone or sequence in a complex
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mixture of clones. The 3-dimensional pooling scheme we used results in superpools that contain a maximum of 768 clones each (assuming no empty wells), or about 1 6 of a genome-equivalent. Contrasting the genome size of AB1380 of about 14 Mb to the average insert size of 736 kb, any given YAC insert sequence would be present only once in a background of 11,316 Mb of total DNA (10,752 Mb of yeast chromosomes, 0.564 Mb of YACs), resulting, effectively, in a mass ratio about four times less advantageous than the mammalian genome. This raises concern about the proper representation of, and ability to find, sequences of interest. Using 48 different rat microsatellite primer pairs, we were gratified to find an average of 9.3 positive, specific signals per primer pair, which again is in keeping with our calculated size and representation of the library (Fig. 3). The well-appreciated avidity for homologous recombination in the yeast genome, along with coligation or cotransformation events during the library construction process, makes chimeric clones and clones with deletions or other rearrangements a frequently observed phenomenon in all YAC libraries. Thus, in most libraries constructed to date about one-third to onehalf of all clones are chimeric. While recombinationdeficient strains of Saccharomyces cerevisiae have been available for some time, their transformation efficiency remains poor, and no large library has been constructed in any of these strains so far; therefore, we used the conventional AB1380 strain despite its disadvantages. To assess the proportion of chimeric clones present in our library we performed FISH analysis on 39 randomly chosen YACs (from among those that yielded only a single band on Southern analysis with total rat DNA). Of those, 8 showed clear evidence of chimerism with pairs of signals on different homologous chromo-
FIG. 2. Library screening by hybridization. Two probes were pooled and used to screen the entire library (three 22 1 22 cm filters, one of which is shown). Positive clones could be identified by the characteristic double signal within one 4 1 4 dot array.
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FIG. 3. PCR-pool screening of the rat YAC library for clones containing the microsatellite marker D1Mgh7. (Top) Agarose gel analysis of PCR amplification of 67 secondary pools. Two negative controls (host yeast genomic DNA in the left-most lane of the upper row, and no DNA in the right-most lane, upper row) and two positive controls (total rat genomic DNA in the second lane from the left in both upper and lower row of wells). Nine positive secondary pools for this marker are found. (Bottom) Agarose gel analysis of 28 primary DNA pools representing one of the positive secondary pools. Left-most and right-most lanes contain a 100-bp molecular weight marker, lanes 2 –9 (from the left) represent the 8 plate pools, lanes 10–17 represent the 8 row-pools, and lane 18 –29 represent the 12 column pools. Lane 30 contains a positive control (total rat genomic DNA) and lane 31, the respective secondary pool represented in this analysis. From the amplification pattern the address of the positive clone is plate 1, row 5, and column 5.
somes. Four YACs produced multiple (more than 4) hybridization signals at chromosomal sites known to contain repeat DNA sequences and are thus likely to carry satellite DNA or rDNA sequences, whereas the remaining 27 YACs produced only a single pair of signals on homologous chromosomes (Fig. 4). These results place our library right within the range of about 20–30% chimerism that would be predicted by past experience. DOP-PCR has been demonstrated to produce signals more uniformly along the chromosomes than interspersed repetitive sequence PCR (IRS-PCR); thus the likelihood that we missed chimerism due to biased amplification is low and we believe our estimate to be a realistic one. Genomic libraries are essential tools for the analysis of complex genomes and the discovery of novel genes,
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particularly genes localized by positional cloning. Different cloning capacity, host and vector systems, degree of manipulation possible, and ease or difficulty of construction determine the special features of various cloning systems, usually making different cloning systems advisable for different stages of genome analysis. Among large-insert genomic cloning systems, YACs have been widely used during the initial stage of positional cloning when the resolving power of linkage analysis usually defines only a relatively large window and have proven to be superior tools in the assembly of far-spanning contigs and the construction of physical maps. Since the original description of the technique (Burke et al., 1987), a large number of libraries have been constructed in different species, including human (Albertsen et al., 1990; Anand et al., 1990; Brownstein et al., 1989; Larin et al., 1991; Traver et al., 1989), mouse (Larin et al., 1991; Rossi et al., 1992), Caenorhabditis elegans (Coulson et al., 1988), Drosophila melanogaster (Garza et al., 1989), commercially important agricultural animals such as cattle (Libert et al., 1993) and pig, (Leeb et al., 1995), as well as in plants (tomato, maize, Arabidopsis). A large number of genes owe their detection to the availability of these libraries, despite their drawbacks as noted above, such as low cloning efficiency, chimerism, and difficulty of handling. The ability of YACs to carry, by a wide margin, the largest genomic inserts makes this system still an essential one, particularly for as yet poorly characterized genomes such as the rat. The value of YACs is of course further augmented by the concomitant availability of other, complementary libraries in different cloning systems, such as BACs (Shizuya et al., 1992)
FIG. 4. Fluorescence in situ hybridization of rat metaphase chromosomes for a randomly chosen YAC clone.
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and PACs (Ioannou et al., 1994), which are based on the E. coli F factor and afford high cloning efficiency, a low degree of chimerism, and relative ease of handling (Shizuya et al., 1992; Woo et al., 1994; Cai et al., 1995; Wang et al., 1994; Kim et al., 1996). Generating such libraries and integrating them with the currently created YAC library will be the next important task we face in creating a comprehensive set of genomic resources for the laboratory rat. ACKNOWLEDGMENTS We thank Kevin Benjamin, Sridharam Raghavan, Humphrey Wattanga, and Carl Mark for their technical assistance in arraying the YAC library. This work was supported by a Research Career Development Award (K04-HL03138-01) from the National Heart, Lung, and Blood Institute to K.L.; by Grant GA175/8-1 from the German Research Council (DFG); by Grant 01KW9607 from the German Ministry for Education, Science, Research and Technology (BMBF), and by a grant from the EC Biotech Program. Resource availability. High-density spotted filters are available through the Resource Center/Primary Database (RZPD) of the German Human Genome Project (Website address: http://rzpd.rzberlin.mpg.de/). PCR pools and screening are available through Research Genetics Inc. (AL, USA; Website address: http://www.resgen.com/).
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