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Original Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2007, 39: 591-598
doi:10.1111/j.1745-7270.2007.00317.x

Cloning of the Rabbit HPRT Gene Using the Recombineering System

Jianjun SHI1, Donghui CAI2, Xuejin CHEN3, and Huizheng SHENG3*

1 Program for Graduation Studies, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;

2 School of Life Science, Soochow University, Suzhou 215006, China;

3 Center for Developmental Biology, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200092, China

Received: April 2, 2007�������

Accepted: April 30, 2007

The study was supported by the grants from the National Basic Research Program of China (001CB509903, 001CB509904), the Hi-Tech� Research and Development Program of China (2001AA216121, 2004AA205010), the National Natural Science Foundation of China (30040003), Science and Technology Committee of Shanghai Municipality (99DJ14002, 00DJ1 4033, 01DJ14003, 03DJ14017), the Chinese� Academy of Sciences (KSCX-2-3-08)

*Corresponding author: Tel, 86-21-55570361; Fax, 86-21-55570017; E-mail, [email protected]

Abstract������� Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.

Keywords������� hypoxanthine phosphoribosyltransferase; BAC; gap repairing vector; genomic organization; homologous recombination

Hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8), an enzyme that catalyzes the conversion of hypoxanthine� and guanine to their respective 5'-mononucleotides, is essential for the metabolic salvage of purines in mammalian cells. A deficiency of the enzyme causes the clinical disorders of Lesch-Nyhan syndrome and gouty arthritis in human males [1]. In mammalian cells, the x-linked HPRT gene has been extensively used in mutation� studies because of its functional haploidy. It is used to design powerful selections for isolating cells lacking� enzyme activity. The orthologs of the HPRT gene have been cloned in mouse [2] and human [3], and the gene抯 exon-intron organization is conserved in these mammalian species. Gene targeting at the HPRT locus has successfully� corrected a mutant HPRT gene in mouse embryonic stem (ES) cells [4]. Importantly, the HPRT locus has been used as an optimal surrogate site for integrating a copy of a transgene into the genome by a precise homologous recombination� event [5,6].

To obtain an animal model for Lesch-Nyhan syndrome, two groups independently reported success in generating HPRT-deficient male mice. But they did not find any spontaneous� behavioral abnormalities characteristic of Lesch-Nyhan syndrome in these mice [7,8]. In 1996, Engle et al. obtained HPRT/adenine phosphoribosyltransferase (APRT) doubly deficient mice, but they did not observe any behavioral abnormalities related to Lesch-Nyhan syndrome� in humans [9]. Until now, there has been no animal model for Lesch-Nyhan syndrome.

Rabbit ES cells represent an excellent in vitro system to study gene expression and regulation in stem cell self-renewal� and differentiation. They are also a potential resource� for producing transgenic rabbits by somatic cell nuclear transplant and gene targeting. Transgenic rabbits� provide a great advantage compared to transgenic mice because, as a relatively large mammalian model, they has provided unprecedented opportunities to study human disease� mechanisms and alternative ways to produce therapeutic� proteins to these diseases [10,11]. Rabbit ES cells have been isolated [12,13] and can proliferate for a prolonged period in vitro while remaining pluripotent. They readily integrate and express exogenous genes and can be used as nuclear donors to generate cloned rabbits [12,14].

To facilitate gene targeting in rabbit ES cells and the production of transgenic rabbits, we cloned and analyzed the rabbit HPRT gene and its cDNA.

Materials and Methods

Bacterial strain

Recombinogenic strains EL350 that carry a defective l prophage with inducible Red recombination proteins were kindly provided by Dr. Neal Copeland (National Cancer� Institute-Frederick, Frederick, USA) [15].

Rabbit bacterial artificial chromosome (BAC) library screening

Rabbit BACs were obtained from the Children's Hospital� Oakland Research Institute (Oakland, USA). The BAC clones containing the rabbit HPRT gene were isolated by screening an LBNL-1 rabbit BAC library with genomic-specific� overgo probes followed by polymerase chain reaction� (PCR) identification. The overgo probes (Hprt-Ova, 5'-ATTGTAGCCCTCTGTGTGCTCAAG-3'; and Hprt-Ovb, 5'-AGAACTTATAGCCCCCCTTGAGCA-3') and PCR primers (OCHprt7, 5'-CCCTCGAAGTGTTG�GAT��ACAGG-3'; and OCHprt8, 5'-GTCAAGGGCATA�TCC��TAC�AACAAAC-3') were designed based on the partial� cDNA sequence of the rabbit HPRT gene (GenBank accession� No. AF020294).

In silico cloning of rabbit HPRT cDNA

A BLASTN (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides&�PROGRAM�=blastn&�MEGA�BLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYPE
=BlastSearch&SHOW_DEFAULTS=on) search of the rabbit expressed sequence tags database (dbEST) using a partial cDNA sequence of the rabbit HPRT gene as a query identified three EST sequences (GenBank accession Nos. EB373457, EB373458 and EB378005). These EST sequences� were retrieved, assembled into a contig sequence, and used to guide the isolation of the gene and its cDNA. DNase I-treated total RNA from the adult liver tissue was reverse-transcribed using M-MLV reverse transcriptase� (Promega, Madison USA) and a putative gene-specific primer (HPRT-RT, 5'-TGGTAATTT�ATTTGATTGCA-3'). A fragment containing the complete coding region was then amplified using the sense primer [5'-GAGCGAGC�CTC�TCGGCTTTCC-3', located in the putative 5' untranslated region (UTR)] and the anti-sense primer (5'-ATTCAATCACTTCTGTTCTTTCCTG-3', located� in the putative 3' UTR). All of these primers were designed according to the assembled contig sequence.

Expression analysis using reverse transcription (RT)-PCR

Total RNA from adult tissues, including heart, liver, spleen, kidney, brain and muscle, was isolated and reverse�-transcribed using random hexadeoxyribonucleotide primers� (TaKaRa, Dalian, China). HPRT mRNA was analyzed� by RT-PCR using primers 5'-GTAATCGGTGG�AGATGA�TCTCTCA-3' and 5'-GTCAAGGGCATATCC�TA�CAA�CA�AAC-3'. Water was used as the negative control.

Cloning the entire HPRT gene

The assembled contig sequence was used as a query to search the rabbit whole genome shotgun sequences database using the BLASTN tool. As shown in Fig. 1(A), five sequences were identified (GenBank accession Nos. AAGW01580029, AAGW01067486, AAGW01700953, AAGW01715926 and AAGW01580025). The approach to clone the complete genomic DNA of the HPRT gene is shown in Fig. 1(B). Four overlapping DNA fragments spanning the entire gene were subcloned by homologous recombination, sequenced and assembled.

Construction of gap repairing vectors

An HpaI restriction site was introduced into the downstream region of the Ap resistance cassette in the pKS- plasmid by PCR amplification using primers 5'-AGTTAACATTTCCCCGAAAAGTGCCAC-3' (HpaI restriction� site underlined) and 5'-ACTCCGCTCATGA�GAC�AATAACCCTG-3' (GenBank accession No. X52329). The modified vector (mpKS) maintains all the characteristics� of pKS-plasmid. The vector was linearized with HpaI or EcoRV, and the blunt ends were modified by a T-tailing procedure [16].

A three-step procedure was carried out to synthesize the linear gap repairing vector (Fig. 2). First, four upstream homology arms (F1R1, F3R3, F5R5 and F7R7) were amplified from BAC DNA and inserted into the HpaI site of mpKS plasmid by TA cloning. Second, four downstream homology arms (F2R2, F4R4, F6R6 and F8R8) were amplified from BAC DNA and inserted into the EcoRV site of four recombinant plasmids (mpKSF1R1, mpKSF3R3, mpKSF5R5 and mpKSF7R7) by TA cloning, respectively. Finally, the four recombinant plasmids (mpKSF1R1F2R2, mpKSF3R3F4R4, mpKSF5R5F6R6 and mpKSF7R7F8R8) were used to prepare the four linear gap repairing vectors by PCR using the primers shown in Table 1.

Transformation of BAC into EL350 recombinogenic strains

A single colony of Escherichia coli DH10B containing 304M19 BAC was grown overnight in 5 ml of Luria broth (LB) with chloramphenicol (12.5 mg/ml). BAC DNA was isolated according to the BAC miniprep protocol using the Plasmid Mini kit (Qiagen, Valencia, USA). A single colony of EL350 cells was inoculated in 5 ml LB at 32 �C overnight with shaking. The cells were collected by centrifuging at 3000 g (0 �C) for 5 min the next day. The pellets were resuspended in 900 ml of ice-cold water, transferred to a 1.5 ml Eppendorf tube on ice, centrifuged at 20,000 g (4 �C) for 20 s, and the supernatant was discarded. The washing process was repeated three times, the cells were resuspended in 50 ml of ice-cold water, and mixed with 1 ml (50-100 ng) of freshly prepared� BAC DNA. The DNA-bacteria mixture was transferred into a 0.1 cm cuvette (Bio-Rad, Hercules, USA) and electroporated at 1.8 kV, 25 mF and 200� using a Gene Pulser II electroporator (Bio-Rad, Hercules, USA). One microliter of LB was added to the electroporated bacteria, incubated� at 32 �C for 1 h with shaking, spun down, spread onto a plate containing chloramphenicol (12.5 mg/ml), and incubated for 24 h at 32 �C.

Homologous recombination

A single colony of EL350 containing BAC was inoculated into 5 ml LB with chloramphenicol and incubated at 32 �C overnight with shaking. The next day, 1 ml of the culture was transferred to 20 ml LB with chloramphenicol and incubated at 32 �C for approximately 2 h (A600=0.5) with shaking. The culture (10 ml) was transferred to a 50 ml Falcon tube and shaken in a 42 �C water bath for 15 min. The tube was immediately put into wet ice, shaken for 2-3 min to make sure that the temperature dropped as quickly as possible, then left in ice for 6 min. Cells were spun at 3000 g (0 �C) for 5 min. The pellet was resuspended in 900 ml of ice-cold water followed by three washes with ice-cold water, as described above. Finally, the cells were resuspended in 50 ml ice-cold water, mixed with 2 ml (200-400 ng) of purified PCR fragments of the four linear gap repairing vectors (mpKSF1R1F2R2, mpKSF3R3F4R4, mpKSF5R5F6R6 and mpKSF7R7F8R8), and electroporated as described above. After electroporation, cells were resuspended in 1 ml LB, incubated at 32 �C with gentle shaking for 1 h, spread onto a plate with ampicillin (60 mg/ml), and incubated at 32 �C for 18-20 h. The recombinant BAC subclones from four linear gap repairing vectors were identified by PCR using sense primers F3, F5, F7, F9 and antisense primer M13R (Table 1).

Results

cDNA cloning of HPRT

Three overlapping EST sequences (GenBank accession Nos. EB373457, EB373458 and EB378005) were identified by searching the rabbit dbEST database using a partial cDNA sequence (GenBank accession No. AF020294) of the rabbit HPRT gene as a query. A 1449 bp contig sequence was assembled and identified by amplifying a 1179 bp cDNA fragment from the liver cDNA pool using a pair of specific primers designed according to the contig (Fig. 3).

The cDNA contains a 154 bp 5'-UTR, a 657 bp open reading frame encoding a protein of 218 amino acids, and a 638 bp 3'-UTR. The nucleotide sequence has been submitted to the GenBank databases under the accession No. EF062857 (Fig. 4). The coding area showed 98%, 97%, 98% and 94% identity in the amino acid sequence with human, mouse, pig and cattle HPRT genes, respectively (Fig. 5), suggesting that this is the rabbit homolog of the HPRT gene.

mRNA expression in adult tissues

Expression of the HPRT gene in the adult rabbit heart, liver, spleen, kidney, brain, and muscle was examined using RT-PCR. A 237 bp PCR product was amplified from cDNAs of adult tissues examined (Fig. 6). RT-PCR analysis showed that the rabbit HPRT gene is expressed ubiquitously in adult tissues.

Cloning the HPRT gene

Five genomic DNA fragments (HPRT-A, HPRT-B, HPRT-C, HPRT-D and HPRT-E) of the putative HPRT gene were obtained through a homology search of the rabbit whole genome shotgun sequences database using the HPRT cDNA sequence as a query. HPRT-A contains exon 1 and partial intron 1. HPRT-B contains partial intron� 1, intron 2, partial intron 3, exon 2 and exon 3. HPRT-C contains partial intron 3, partial intron 4 and exon 4. HPRT-D contains partial intron 5, partial intron 6 and exon 6. HPRT-E contains partial intron 6, intron 7, intron 8, exon 7 and exon 9. The relative positions of the five fragments in the HPRT gene are shown in Fig. 1(A).

As well as the sequenced area, the sequences of four big gap regions are still missing. The genomic DNA of these big gap regions was subcloned from the BAC clone 304M19 using a recombineering-based method. The 304M19 clone contains the complete coding region of the HPRT gene. The subclones were PCR screened and those with PCR products of the expected sizes were selected and sequenced. Four fragments, approximately 12, 13, 10.5 and 14.5 kb in length, were obtained, which covered all the missing gaps in the HPRT gene [Fig. 1(B)]. These sequences were assembled into a contig sequence spanning� the complete coding region of the rabbit HPRT gene.

Genomic organization of the rabbit HPRT gene

The assembled sequence of the rabbit HPRT gene has been submitted to the GenBank database under the accession� No. EF219063. Alignment of the HPRT cDNA with the genomic sequence revealed that the entire gene is 47.9 kb in length and split into nine exons. Exon-intron junctions were deduced by comparing the HPRT cDNA with the genomic sequence following consensual splicing signal rules (GT/AG) [17]. The boundary of each exon and its flanking intron sequences are shown in Table 2. The exon-intron organization is similar to that previously determined for the HPRT orthologs of mouse [2,3]. The sizes of the seven internal exons are identical to those of the mouse and human HPRT gene. The 5' end of the gene contains extremely GC-rich sequences and two GC hexanucleotide motifs (5'-GGCGGG-3'), but lacks the prototypical 5' transcriptional regulatory sequence elements. These structural features of the gene are highly conserved.

Discussion

Whole genome shotgun sequencing and ESTs have produced� a tremendous amount of sequencing information� for genes of many species, now available in the public domain. However, sequences generated by these approaches� are often fragmented, small in size (<20 kb), and separated by big gaps of unknown sequences. For complicated large genes, filling in these big gaps could be difficult. The human PDE11A gene is in a genomic DNA region of over 300 kb and contains 23 exons. To obtain its exon-intron organization, long-distance PCR and the screening of the human genomic DNA phage library and BAC library were carried out [18]. For some genes with big introns and small exons, it is impossible to obtain complete� sequences by the PCR method alone.

In order to obtain sequences for the entire HPRT gene, we tried to clone the big gap regions using long-distance PCR. However, complicated template structures in the genome posed problems that were difficult to overcome. In addition, nucleotide substitutions arising from misincorporation by Taq DNA polymerase potentially reduced� the quality of the cloned sequences. The attempt to directly sequence BAC clones by a shotgun sequencing� strategy was time-consuming, laborious and expensive, and generated many superfluous sequencing reactions.

During the past few years, it has become possible to manipulate BAC clones by recombineering in E. coli. Efficient� homologous recombination, mediated by the Red proteins of l phage in E. coli, permits insertion of linear fragments into the BAC constructs, as well as the subcloning of DNA fragments from them [19-21]. The gap repairing process that enables homologous recombination between the linear gap repairing vector and the genomic� DNA in the BAC clones makes it very convenient� to subclone large-sized DNA from the BAC constructs into high-copy plasmid vectors.

We have used sequences provided by databases to design arms� to subclone large DNA sequences from the BAC clones through homologous recombination. The approach, which combines bioinformatics with recombineering, has greatly improved the efficiency of subcloning and sequencing� of the rabbit HPRT gene. Our work showed that this approach is very efficient, and should be applicable to similar works on large-sized genomic DNA.

Acknowledgement

We are grateful to Dr. Neal Copeland for kindly providing� a recombineering system.

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