Radial Transformation-Associated Recombination Cloning from the Mouse Genome: Isolation of Tg.AC Transgene with Flanking DNAs

Genomics 70, 292–299 (2000) doi:10.1006/geno.2000.6384, available online at http://www.idealibrary.com on

Radial Transformation-Associated Recombination Cloning from the Mouse Genome: Isolation of Tg.AC Transgene with Flanking DNAs Michael C. Humble,* ,† Natalya Kouprina,† Vladimir N. Noskov,‡ Joan Graves,‡ Ed Garner,† Raymond W. Tennant,† Michael A. Resnick,‡ Vladimir Larionov,‡ and Ronald E. Cannon† ,1 *Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514; and †Laboratory of Environmental Carcinogenesis and Mutagenesis and ‡Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, 111 Alexander Drive, Research Triangle Park, North Carolina 27709 Received June 9, 2000; accepted September 14, 2000

Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA. We report the first example of radial TAR cloning from the mouse genome. Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene. Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11. Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression. YACs containing variable lengths of 5ⴕ or 3ⴕ flanking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line. A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones. All Tg.AC BACs expressed the transgene in this in vitro system. This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene’s integration site. © 2000 Academic Press

INTRODUCTION

Traditional methods for the isolation of specific chromosomal regions from complex genomes are laborious and require the construction and screening of large libraries of clones. Transformation-associated recombination (TAR) cloning is a novel approach that allows entire genes and large chromosomal regions to be spe1

To whom correspondence should be addressed at National Institute of Environmental Health Sciences, P.O. Box 12233, MD F1-05, 111 Alexander Drive, Research Triangle Park, NC 27709. Telephone: (919) 541-3821. Fax: (919) 541-1460. E-mail: [email protected].

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cifically, accurately, and quickly isolated from total genomic DNA (Larionov, 1999). This nonenzymatic procedure for gene cloning, previously described for the isolation of the human BRCA1, BRCA2, and HPRT genes (Larionov et al., 1997; Kouprina et al., 1998), is carried out utilizing the yeast Saccharomyces cerevisiae, an organism that exhibits a high level of intermolecular recombination between homologous DNAs during transformation. Gene cloning is based on the cotransformation of yeast spheroplasts with a DNA vector containing two homologous sequence regions (“hooks”) and gently isolated mammalian genomic DNA. In radial TAR cloning, one hook is specific to the gene of interest, while the other hook is specific for an interspersed repetitive element, such as the Alu sequences in humans. Recombination between the radial TAR cloning vector and genomic DNA results in the generation of various size circular yeast artificial chromosomes (YACs) extending from the gene-specific sequence to different upstream repetitive positions. This report represents the first successful gene isolation by radial TAR cloning from the mouse genome. In this study, radial TAR cloning was applied for the selective cloning of the Tg.AC transgene cassette from the Tg.AC transgenic mouse. Created on the FVB/N mouse, genetically initiated Tg.AC transgenic mice carry a v-Ha-ras transgene regulated 5⬘ by a 1000-bp sequence corresponding to the basal promoter of the mouse zeta-globin gene and flanked 3⬘ by an SV-40 polyadenylation sequence (Leder et al., 1990). Tg.AC transgene expression cannot be detected in normal skin, but can be detected in the skin following full thickness wounding or exposure to UV light or chemical carcinogens (Leder et al., 1990; Cardiff et al., 1993; Hansen et al., 1996). Though the inductive mechanism is not understood, previous studies (Leder et al., 1990; Cardiff et al., 1993; Hansen and Tennant, 1994a,b; Hansen et al., 1996; Cannon et al., 1997, 1998) support the belief that the transgene becomes activated in response to promotional stimuli as a downstream event in the tumorigenic mechanism. Due to its discriminate

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tumorigenic response to promotional stimuli (Leder et al., 1990; Spalding et al., 1993), the Tg.AC mouse is currently under investigation as a short-term screen for carcinogens. Our goal is to determine the molecular mechanisms regulating the induction and sustained expression of the oncogenic v-Ha-ras transgene. Since only one of four founder lines displayed the unique characteristics now associated with the Tg.AC mouse (Leder et al., 1990), we hypothesized that Tg.AC transgene expression was enabled or dependent upon an upstream or downstream flanking control sequence found near the transgene integration site on chromosome 11, a relationship similar to those observed between the endogenous ␣- and ␤-globin gene loci and their respective locus control regions (Grosveld et al., 1987; Higgs et al., 1990). To explore the possible presence of a flanking control region, radial TAR cloning (Kouprina et al., 1998) was applied to isolate the Tg.AC transgene and flanking chromosome 11 regions. A vector containing the Tg.AC’s unique SV-40 sequence and the 130-bp mouse B1 repetitive element was utilized to construct orientation-specific YACs of varying sizes containing a portion of the multicopy Tg.AC transgene array and flanking mouse chromosomal DNA. Following conversion of YACs to bacterial artificial chromosomes (BACs), the BACs are transfected into an FVB/N carcinoma cell line and assayed for their ability to promote transcription of the transgene. MATERIALS AND METHODS Fluorescence in situ hybridization (FISH). FISH was performed as previously described (Afshari et al., 1994). The SV-40 region of the Tg.AC transgene and the BRCA1 gene from a p1 plasmid (compliments of Roger Wiseman, National Institute of Environmental Health Sciences) were labeled with fluoresceinated dUTP (Catalog No. US77137, Amersham Pharmacia Biotech Inc., Piscataway, NJ) by nick-translation using a kit purchased from GibcoBRL Life Technologies (Gaithersburg, MD). Construction of TAR cloning vector. The TAR cloning vector was constructed by ligation of an SV-40 fragment into a ClaI–SalI site adjacent to the B1 site of vector pVC-B1 (B1–CEN6 –HIS3–AmpR). The SV-40 fragments were constructed by polymerase chain reaction (PCR) using primers containing overhanging ClaI and SalI sequences, in addition to the SV-40 sequences. Through the use of matched sets of Cla/Sal primers, an SV-40 amplicon was produced that, when utilized in TAR cloning vectors, will result in YAC formation containing flanking chromosome 11 in a specific orientation to the Tg.AC transgene. The matched set of primers was as follows: SV-40 Sal Rev (5⬘-GCGCGTCGACCTTGTATAGCAGTGCA-3⬘), and SV-40 Cla For (5⬘-GCGCATCGATGACAAACTACCTACAG-3⬘); and SV-40 Sal For (5⬘-GCGCGTCGACGACAAACTACCTACAG-3⬘), and SV-40 Cla Rev (5⬘-GCGCATCGATCTTGTATAGCAGTGCA-3⬘). PCR was performed on a full-length pGEM 3/Tg.AC construct (obtained from the Leder laboratory) (Leder et al., 1990) as a template at 4 mM MgCl 2. The amplification protocol consisted of a 1-min denaturation at 94°C, 3 cycles of a 30-s denaturation at 94°C, a 30-s annealing at 42°C, and a 30-s extension at 72°C, followed by 30 cycles of a 30-s denaturation at 94°C and a 1-min extension at 72°C. PCR products were phenol:chloroform extracted and precipitated by adding 1/10 vol of 3 M sodium acetate, 2 vol of 100% ethanol. ClaI and SalI restrictions were set up for the pVC-B1 vector and the PCR products, and then the products were phenol:chloroform extracted, precipi-

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tated with sodium acetate– ethanol, and ligated overnight at room temperature. Competent cells were transformed with the TAR cloning vectors, and plasmid isolations were performed with Qiagen Plasmid Purification Kits (see below). Yeast strain and spheroplast transformation. A Saccharomyces cerevisiae strain, VL6-48, with the HIS3 marker deleted (MATa, his3-⌬200, trp1-⌬1, ura3-52, met14, lys2, ade2-101) was used as a host for TAR cloning (Larionov et al., 1996). A protocol for spheroplasting cells that results in efficient transformation was used (Kouprina and Larionov, 1999). The constructed TAR cloning vectors, pVC-B1/4 and pVC-B1/30, were cut with SalI (the site is located between the hooks) before transformation to yield linear molecules bounded by the SV-40 and B1 hooks. Agarose plugs (100 ␮l) containing approximately 5 ␮g of gently prepared DNA from the liver tissue of the Tg.AC mouse along with the linearized TAR cloning vector were presented to the spheroplasts. Yeast transformants were selected on synthetic complete synthetic medium plates lacking histidine. The SV-40 hook lacked yeast ARS activity based on yeast transformation experiments. PCR screening. A pair of primers was utilized for PCR screening of YAC pools and individual transformants: ZGFor (5⬘-GTG AGA GGA ATT ACT GCT TCC-3⬘) and ZGRev (5⬘-AGG CTG CGC TGG AGT TGA GT-3⬘) are specific for the zeta-globin promoter. A PCR product of 419 bp was diagnostic for recombination between the TAR vector and a specific region of the SV-40 terminator present in the cassette. Yeast genomic DNA isolated from the transformants was amplified by using primers under the following standard PCR conditions: 50 mM KCl, 10 mM Tris–HCl, pH 9.0, 3.0 mM MgCl 2, 0.2 mM dTTP, dCTP, dGTP, and dATP in a final volume of 50 ␮l. Thermocycling conditions consisted of 35 cycles of 1 min at 94°C, 45 s at 55°C, and 2 min at 68°C, followed by one cycle of a 10-min extension at 72°C in a 9600 Thermocycler (Perkin–Elmer). Characterization of YAC clones. Chromosome size DNAs from yeast transformants were separated by transverse alternating field electrophoresis (TAFE), blotted, and hybridized with a zeta-globin promoter probe. To estimate the size of circular YACs, agarose DNA plugs prepared from yeast transformants were exposed to a low dose of gamma rays [30 krad (300 Gy)] before TAFE analysis. At this dose, the molecules receive only a single break, and the leading band reveals the size of YACs (Larionov et al., 1996). Retrofitting of YACs into BACs for propagation in bacterial and mouse cells. Retrofitting of YACs into BACs was accomplished through the use of a yeast– bacteria–mammalian cell shuttle vector, BRV1, containing the F-factor origin of replication and the Neo R gene (Kouprina et al., 1998), by a standard lithium acetate transformation procedure. The retrofitted YACs were moved to Escherichia coli by electroporation utilizing electroporation-competent Sure E. coli (Catalog No. 200227, Stratagene, La Jolla, CA), a strain that provides gene stability by limiting rearrangement events on inverted repeats. BAC plasmid isolation. BAC plasmids were isolated from E. coli utilizing a Qiagen Plasmid Purification kit (Catalog No. 12163, Qiagen Inc., Santa Clarita, CA) (Birnboim and Doly, 1979; Kado and Liu, 1981) with slight modification to the manufacturer’s protocol. BAC concentrations were determined on a Hoefer DyNaQuant 200 Fluorometer or on a Beckman DU-600 spectrophotometer. Transfections and cell medium. BAC transfections of adherent FVB/N cells were performed utilizing Lipofectamine (Catalog No. 18324-012, Gibco BRL Life Technologies) following the manufacturer’s protocol. Cells were maintained at 10% CO 2 in medium containing Dulbecco’s modified Eagle’s medium:RPMI (1:1) with 20% fetal bovine serum (FBS) (Gibco BRL), 1 mM glutamine (Gibco BRL), 50 U/ml penicillin (Gibco BRL) and 50 mg/ml streptomycin (Gibco BRL). FBS was heat-inactivated at 56°C for 30 min prior to use. All medium was passed through a 0.45-␮m filter. Transfected cell lines and colonies were selected for and maintained in the above medium additionally containing 500 ␮g/ml G418 (Gibco BRL). Ten G418resistant colonies were selected for each BAC transfection.

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FIG. 1. Chromosomal localization of the Tg.AC transgene. (A) A Tg.AC metaphase spread hybridized with the Tg.AC SV-40 sequence indicates a single locus for the transgene array. The signals are located proximal to the centromere on chromosome 11. (B) A Tg.AC metaphase spread cohybidized with BRCA1 and Tg.AC SV-40 sequences. BRCA1 signals are located on the telomeric ends of chromosome 11. (C) Map of mouse chromosome 11 indicating location of BRCA1 and additional genes (from Mouse Genome Informatics, The Jackson Laboratory, http://www.informatics.jax.org). Isolation and quantitation of cell culture RNA and DNA. To examine the expression of the Tg.AC transgene, total RNA was isolated from cell cultures. RNA was extracted utilizing TriReagent (Molecular Research Center, Cincinnati, OH) following the manufacturer’s protocol (Chomczynski and Sacchi, 1987; Chomczynski, 1993). DNA was extracted utilizing DNAzol (Molecular Research Center) following the manufacturer’s protocol (Chomczynski et al., 1997). RNA and DNA were quantified on a Beckman DU 600 spectrophotometer prior to use. Transgene PCR and reverse transcription-polymerase chain reaction (RT-PCR). The presence of the Tg.AC transgene was determined by PCR amplification as previously described (Humble et al., 1998). To examine the expression of the Tg.AC transgene, total RNA was isolated from cell cultures, and transgene RNA was detected by RT-PCR (Humble et al., 1998). Amplification products were visualized on 2% agarose gels stained with ethidium bromide and photographed with Polaroid type 57 high-speed film. Transgene amplification yields a product of 279 bp for genomic DNA and a 214-bp product for RNA. As a positive control, primers for mouse betaglobulin-2 (MB2) generate a product ⬃217 bp in length for RNA. Restriction analysis of YAC/BACs. Restrictions analysis was performed on BAC DNA as follows: 10 ␮g BAC DNA was incubated with 4 units of enzyme per 1 ␮g DNA in 1⫻ buffer overnight at 37°C. Products were precipitated and loaded onto a 1.2% agarose gel in 1⫻ TBE (0.09 M Tris– borate, 0.002 M EDTA). DNA was transferred to a nylon membrane (Duralon UV Membrane, Stratagene Catalog No. 420104) by overnight capillary action. The transferred DNA was crosslinked (UV Stratalinker 2400, Stratagene) to the nylon membrane, which was then rinsed briefly in distilled H 2O. Nonisotope Southern analysis of YAC/BACs. Nonisotope Southern analyses (Kantz et al., 1999) of BAC DNA restriction digestions were by performed utilizing fluorescein-11– dUTP-labeled probes (Kantz et al., 1999) created according to the protocol from the Gene Images random prime labeling module (Catalog No. RPN 3541, Amersham International plc, Buckinghamshire, England) and the protocol and components from the Gene Images CDP-Star detection module (Catalog No. RPN 3511, Amersham International plc). Isolation of YAC ends by plasmid rescue and DNA sequencing. The YAC ends were isolated as previously described (Bates, 1996). This procedure utilizes the bacterial origin of replication and ampicillin-resistance gene of the pVC-B1 plasmid (B1–CEN6 –HIS3– AmpR) to rescue YAC end clones. DNA from yeast containing YAC clones was digested with an EcoRI restriction endonuclease that is not present in the plasmid. The digested yeast DNAs were then

circularized by ligation and transformed into E. coli by electroporation. Selection for ampicillin resistance was utilized to identify the transformants. Following restriction and ligation, the resultant plasmids contained the original plasmid with a portion of the YAC DNA inserts. The rescued plasmids were checked for the absence of inserts by EcoRI restriction again and then submitted for further sequence analysis. DNA sequencing was performed using a dRhodamine Dye Terminator Cycle Sequencing Kit (Perkin–Elmer, Catalog No. 403 042) in conjunction with a Model 377 automated DNA sequencer (Perkin–Elmer).

RESULTS AND DISCUSSION

This work represents the successful attempt to isolate the Tg.AC transgene and its associated flanking chromosomal regions, with the ultimate goal of identifying and mechanistically understanding the cis region requirements for the tumorigenic response. The Tg.AC transgene is found as a multicopy integrate consisting of approximately 40 copies per genome in hemizygous mice (Thompson et al., 1998). At 3.8 kb per copy, the transgene array encompasses approximately 152 kb. Through FISH, the Tg.AC transgene array was located as a single site of integration proximal to the centromere on chromosome 11 (Fig. 1A). A cohybridization with a BRCA1 probe confirmed the identification of chromosome 11 (Fig. 1B). As a single site of integration, in vivo expression of the transgene following promotional stimuli must be due to the transgene copies found within this locus and not be due to a single copy of the transgene found elsewhere within the genome. Isolation of the trangene array (or a portion thereof) through radial TAR cloning ensures the coisolation of any cis-regulatory regions involved in transgene expression. Isolated YAC/BACs can then be utilized as expression vectors in transfection experiments exploring the requirement of cis-regulatory regions. A radial TAR cloning strategy was devised to allow the isolation of a set of circular YACs containing the

RADIAL TAR CLONING FROM THE Tg.AC MOUSE

FIG. 2. Transgene construct in v-Ha-ras Tg.AC transgenic mice and construction of TAR cloning vectors. (A) Tg.AC transgene construct. SV-40 fragments were constructed by polymerase chain reaction utilizing matched sets of SV-40 primers containing overhanging ClaI and SalI sequences. YAC formation containing flanking regions of chromosome 11 in a specific orientation to the Tg.AC transgene can be achieved following ligation of the SV-40 amplicons into a TAR cloning vector. (B) Schemes of SV-40/B1 TAR cloning vectors, pVCB1/4 and pVC-B1/30, indicating orientation of the SV-40 sequence. The TAR cloning vectors were constructed by ligation of an SV-40 fragment into the ClaI–SalI sites adjacent to the B1 repeat of the vector pVC-B1 (B1–CEN6 –HIS3–AmpR).

v-Ha-ras transgene and nested overlapping fragments extending from the SV-40 terminator portion of the transgene to different upstream or downstream B1 positions in the flanking regions of chromosome 11

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(Kouprina et al., 1998). For this purpose, two TAR cloning vectors, pVC-B1/4 and pVC-B1/30, were generated containing a centromere (CEN6), a yeast selectable marker (HIS3), an ampicillin-resistance gene, a mouse B1 sequence, and an orientation-specific SV-40 sequence (Fig. 2). When the plasmids are linearized, one end contains 373 bp of the SV-40 sequence with the other end containing 130 bp of the B1 repeat (see Materials and Methods). Homologous recombination between Tg.AC mouse DNA and the linearized plasmid results in the formation of circular YACs containing the Tg.AC transgene and flanking mouse chromosome 11 regions (Fig. 3). Six transformation experiments were carried out with freshly prepared yeast spheroplasts as previously described for TAR cloning of single-copy genes (Larionov et al., 1997; Kouprina et al., 1998). Approximately 800 His ⫹ transformants were obtained for each plasmid construction. (Using 1–3 ␮g of mouse DNA, 1 ␮g of vector, and 2 ⫻ 10 9 spheroplasts, there were approximately 200 –300 transformants per experiment.) To identify transformants containing the Tg.AC transgene and flanking regions, 1600 transformants were combined into 50 pools and screened by PCR for the presence of the transgene zeta-globin promoter. A pair of primers that identified the promoter region of the transgene was utilized (see Materials and Methods). PCR products of the predicted size were obtained for 16 pools. Individual clones from 6 pools were identified and rescreened by PCR. Clones 1, 2, and 3 for the vector pVC-B1/4 and clones 1, 2, and 3 for the vector pVC-B1/30 containing the Tg.AC transgene and flanking regions were utilized for further analysis. Genomic DNAs were isolated from the original transformants and analyzed by TAFE. As expected for circular YACs, the transgene hybridizing material was retained in the starting wells of the gel (data not shown). To estimate the size of the cloned material, agarose plugs containing the YAC DNA were irradiated with a low dose of ionizing radiation. Based on TAFE analysis of the irradiated DNAs, isolates 1, 2, and 3 (plasmid pVC-B1/4) contained circular YACs of 50, 40, and 70 kb in size, and isolates 1, 2 and 3

FIG. 3. Isolation of a series of circular YACs containing the Tg.AC transgene and flanking chromosome 11 regions by radial TAR cloning. Yeast spheroplasts are transformed with genomic Tg.AC mouse DNA along with a TAR cloning vector containing a SV-40 terminator-specific sequence and a B1 repeat at the ends of the linearized plasmid. Recombination between the sequences in the vector and genomic DNA leads to the establishment of circular YACs that extend from the SV-40 sequence to various B1 positions. Relative YAC orientations were deduced from restriction digests and Southern blot hybidization with a B1 probe. Approximate size is indicated under the B1 sequence for each YAC. CEN corresponds to the yeast chromosome VI centromere, and HIS3 is a selectable marker.

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FIG. 4. Tg.AC transgene expression in BAC-transfected FVB/N carcinoma cells. Total RNA from BAC transfected FVB/N colonies was assayed for transgene expression by RT-PCR. Included are RNA samples from a transgene expressing Tg.AC carcinoma cell line (lane Tg), the FVB/N carcinoma cell line (lane F), one colony from each BAC- FVB/N transfection (lanes 1–6), and a water blank (lane ⴚ). (A) Tg.AC transgene RT-PCR assay. (B) RT-PCR for transgene expression. RT(⫹) reactions show the RNA-derived 214-bp product and the DNA-derived 279-bp product. RT(⫺) reactions show the DNAdependent 279-bp product. RT-PCR for housekeeping gene MB2 generates a 217-bp product.

(plasmid pVC-B1/30) contained circular YACs of 100, 70, and 60 kb in size. The YACs, three from each orienting B1/SV-40 TAR cloning vector, were converted to BACs with a mammalian selectable marker using the BRV1 retrofitting vector and then electroporated into E. coli for DNA isolation. Bacterial clones containing independent isolates of the Tg.AC (BAC4-1, BAC4-2, BAC4-3 and BAC30-1, BAC30-2, and BAC30-3) were used for DNA isolation and further analyses. DNAs from the six independent Tg.AC isolates were isolated from bacterial cells and examined by restriction analysis for the presence of full-length copies of the transgene. All of the BACs contained full-length transgene constructs. Based on relative hybridization signals obtained from DNA blots, transgene copy numbers were estimated to be 4, 7, and 10 for BAC4-1, BAC4-2, and BAC4-3 and 10, 7, and 7 for BAC30 -1, BAC30-2, and BAC30-3, respectively. The BACs were transfected into a FVB/N carcinoma cell line by lipofection. Transfected cells were selected for by neomycin (G418) resistance. To detect transcriptional activity of the transgene, RT-PCR assays designed to detect specifically the presence of transgene transcripts were performed on RNA purified from 10 neomycin-resistant colonies from each transfected BAC. All of the colonies from each BAC transfection were shown to enable transgene expression in the FVB/N cell line (Fig. 4), regardless of the relative orientation or size of the flanking regions contained within.

As with any transfection-expression experiment, a number of parameters must be considered when interpreting these results. The cell type utilized, copy number, integration site of the vector, and integrity of the transgene array must all be accounted for in the final analysis. The FVB/N carcinoma cell line utilized in this experiment was derived from a squamous cell carcinoma generated on the back of an FVB/N mouse treated with an initiating dose of 7,12dimethylbenz[␣]anthracene (DMBA) followed by 12-Otetradecanoylphorbol-13-acetate (TPA) promotion. Characterization of this cell line indicated the existence of a codon 61 mutation in the endogenous Ha-ras gene. We felt that with its activated ras, the isogenic FVB/N carcinoma cell line represented an inductive process nearly parallel to that found in the Tg.AC. Transgene copy numbers were estimated for each YAC/ BAC. The RT-PCR assay utilized is not quantitative, so the effect of copy number on expression levels cannot be discerned. Ten stable clones were isolated for each YAC/BAC transfection to ensure that any subsequent expression of the Tg.AC transgene was not due to the YAC/BAC integration site, promoter/enhancer adoption, or copy number. All clones isolated from each transfected YAC/BAC expressed the transgene, thus diminishing the possibility of a position effect. A remote limitation to any system utilizing antibiotic selection entails the unique situation in which a cloned negative regulatory region silences the expression of the selectable marker. Likewise, in our own system, it would be possible that a cis negative regulatory element within the chromosome could be cloned into the YAC/BACs and silence G418 resistance. These YAC/ BACs would not allow stable clones to be isolated in the presence of G418, thus the identification of a negative cis regulatory region could be missed. Maintenance of the intact transgene array within the YAC/BACs has proven to be a complex issue. Subsequent to the onset of this experiment, a discovery by the Sistare laboratory (FDA) (Thompson et al., 1998) provided insight into the arrangement of transgene copies within the Tg.AC transgene array. Within the transgene array there are two copies of the transgene construct in a head-to-head inverted orientation (43). These copies form a palindromic repeat. Since the exact location of this inversion within the transgene array has not been determined, the potential symmetry or asymmetry of the transgene array is not known. The in vivo response of Tg.AC mice to promotional stimuli is dependent upon the presence of a 2.0-kb band representing a head-to-head zeta-globin promoter palindrome from BamHI-digested genomic DNA (Thompson et al., 1998). A deletion as small as 100 bp across the central EcoRI site within the palindrome eliminates both the in vivo expression of the Tg.AC transgene and the subsequent tumorigenic phenotype in a hemizygous Tg.AC mouse. The presence of this inversion had the potential to reverse our orientation directed cloning strategy. To

RADIAL TAR CLONING FROM THE Tg.AC MOUSE

FIG. 5. Southern blot hybridization with a B1 probe of HindIII digested BAC DNAs. BAC DNAs were HindIII digested, electrophoresed on an agarose gel, and transferred to a nylon membrane. The membrane was hybridized with a 128-bp B1 probe. A 1-kb DNA ladder is utilized for size comparison. Distinct 850-bp B1 bands were identified in four of the BACs (A), while 2.3-kb bands were identified in the remaining two BACs (B).

determine the orientation of each YAC/BAC isolate with respect to the transgene, restriction digests and DNA blots were performed. HindIII digestion of the BACs leaves the B1 sequences intact and creates a 2-kb transgene fragment representing adjacent SV-40 and zeta-globin promoter sequences from two transgene copies (Fig. 1). DNA blotting with a B1 probe created a “fingerprint” by which related BACs could be ascertained (Fig. 5). An 850-bp B1 band was identified in four of the BACs, while a 2.3-kb band was found in the remaining two BACs (labeled A and B, respectively, Fig. 5). This result revealed that four of the six BACs analyzed were oriented in one direction on the chromosome (BAC30-1, BAC30 -2, BAC30-3, and BAC4-1), while the other two (BAC4-2 and BAC4-3) were oriented in the opposite direction (Fig. 3). In addition, the characterization of the BACs revealed potential structural information regarding the transgene array. HindIII digestion of the BAC DNAs followed by blot hybridization with the SV-40 probe created unique bands of approximately 3 and 4.2 kb in size (data not shown). These bands supported the relative orientations of the BACs as determined by the B1 probe and are hypothesized to represent fragments containing SV-40 sequences from the outermost transgene copies of the multicopy array and flanking chromosome 11. Bands of this nature were not seen when the HindIII digestions were blotted with the zeta-globin promoter. These results support an arrangement in

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which the transgene copies are oriented outward toward the chromosome from a palindrome arrangement present somewhere among the copies. Due to the nature/construction of the transgene array and the orientation of the SV-40 sequence in the TAR cloning vectors, not all of the original Tg.AC YACs would have been expected to contain this palindrome (Fig. 3). The BAC DNAs were digested with BamHI and probed with the zeta-globin promoter. An intact palindrome arrangement was known to exist in the Tg.AC genomic DNA; however, the 2-kb band representing the head-to-head transgene promoters was absent from the six isolated BACs. A loss of the head-to head palindrome structure during propagation of the cloned materials in E. coli is one possible explanation for its absence in the BACs. Inverted or palindromic DNA sequences are difficult to propagate in E. coli (Collins et al., 1982). Although an intact head-to-head arrangement of zeta-globin promoters is required for the induced expression of the transgene in vivo, its absence from the YAC/BACs did not prevent expression in vitro when transfected into the FVB/N carcinoma cell line. Since the BAC transgene expression was assayed in a carcinoma cell line, one possible explanation may be that factors required for the sustained expression of the Tg.AC transgene, once present, circumvent the requirement of an inductive process that is palindrome dependent. This explanation does not preclude the need for the palindromic arrangement of the transgene to initiate transgene expression in vivo; however, once a tumorigenic potential is achieved, the palindrome is no longer needed. As support for this hypothesis, a Tg.AC carcinoma cell line, derived from TPA-promoted, transgene-expressing papillomas, lost its palindromic zeta-globin promoters over time yet maintained its transgene expression. Transfection of YAC/BACs into a noncarcinoma cell line may demonstrate how a loss of the central palindromic promoter region affects expression of the transgene. Results from such experiments may help explain the observation that nonresponding Tg.AC mice (those with either truncated or absent zeta-globin promoter palindromes) do not express the transgene in papillomas or carcinomas produced by DMBA/TPA treatment. An ideal system for examining transgene expression would be the transfection of palindrome-containing Tg.AC YACs into the skin of FVB/N mice, with a subsequent promotion by the phobol ester, TPA. This system would most closely model the in vivo cascade of events necessary for the induction of transgene expression and the resultant tumorigenic outcome in the Tg.AC mouse. Results from the transfected BACs into the FVB/N carcinoma cell line demonstrate that flanking chromosomal DNA may not be required for sustained transgene expression. Since the BACs contained flanking chromosomal DNA in only one orientation relative to the transgene, the results do not address the possibility that expression or repression occurs in vivo from

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the interaction of both flanking regions with the transgene array. The creation of new YACs containing both upstream and downstream flanking DNA, as well as the 2.0-kb palindromic promoter sequence, would shed light on this potential mechanism of regulation of Tg.AC transgene expression. The potential presence of palindromic copies within a transgene array spawns new considerations when isolating transgene copies from genomic DNA by TAR cloning. Large palindromic sequences may be inherently unstable and susceptible to truncation or elimination from the YAC/BACs during cloning and/or amplification. It is unlikely that the palindromic sequence within the Tg.AC YAC/BACs was excised by yeast, as palindromic sequences that are not clonable in E. coli can be propagated in yeast cells (Lobachev et al., 1998). Care must be taken to maintain palindrome-containing YACs in palindrome-permissive yeast to ensure the integrity of the in vivo construct. Propagation of palindrome-containing YAC/BACs in E. coli should be avoided. A subsequent analysis of new YAC isolates may demonstrate that the Tg.AC promoter palindrome can be isolated and maintained in yeast. Additional experimentation utilizing palindrome-containing YACs can then be performed. Sequence information on the transgene’s flanking chromosomal regions might prove useful in better understanding the molecular mechanism of the Tg.AC mouse response to chemical carcinogens. For this purpose, the YAC ends were rescued as plasmids in E. coli (see Materials and Methods) and sequenced using the chain-termination method. The sequencing data demonstrated that homologous recombination between genomic DNA and the two arms of the TAR cloning vectors occurred since both homologous arms were fully preserved and present in the YAC/plasmid isolates (data not shown). The unique sequences from the flanking regions were compared with all sequences within GenBank using the on-line BLAST service at NCBI. This search failed to detect any significant homology with available sequences. Unique sequences from the flanking regions will be deposited with GenBank, as this sequence information will be useful to the mouse genome project as well as further work in Tg.AC. In this report, we demonstrate that radial TAR cloning of a specific gene is possible from the mouse genome and that the isolated gene can be expressed in vitro. We have also shown that the Tg.AC transgene array is located as a single site of integration proximal to the centromere on chromosome 11. The radial TAR cloning strategy proved to be a quick and useful method for isolating a portion of the Tg.AC transgene array along with large regions of flanking chromosomal DNA and for examining Tg.AC gene expression in mammalian cells. The ability to orient the flanking DNA to the gene of interest by radial TAR cloning should enable future scientists to explore and isolate locus control regions and examine their effect on gene expression.

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