Original Paper
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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? 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‘-CCCTCGAAGTGTTGGATACAGG-3‘; and OCHprt8, 5‘-GTCAAGGGCATATCCTACAACAAAC-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&MEGABLAST=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‘-TGGTAATTTATTTGATTGCA-3‘).
A fragment containing the complete coding region was then amplified using the
sense primer [5‘-GAGCGAGCCTCTCGGCTTTCC-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‘-GTAATCGGTGGAGATGATCTCTCA-3‘
and 5‘-GTCAAGGGCATATCCTACAACAAAC-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‘-ACTCCGCTCATGAGACAATAACCCTG-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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