Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5)
Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined.
A Blast search of mouse genomic databases failed to locate the ESG1 gene. We identified several bacterial artificial clones containing the mouse ESG1 gene and an additional ESG1-like sequence with a similar gene structure from chromosome 9. The ESG1-like sequence contained a multiple critical mutations, indicating that it was a duplicated pseudogene. The 5' flanking region of the ESG1 gene, but not that of the pseudogene, exhibited strong enhancer and promoter activity in undifferentiated ES cells by luciferase reporter assay. To study the physiological functions of the ESG1 gene, we replaced this sequence in ES cells with a β-geo cassette by homologous recombination. Despite specific expression in early embryos and germ cells, ESG1-/- mice developed normally and were fertile. We also generated ESG1-/- ES cells both by a second independent homologous recombination and directly from blastocysts derived from heterozygous intercrosses. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells. These ES cells demonstrated normal morphology, proliferation, and differentiation.
The mouse ESG1 gene, together with a duplicated pseudogene, is located on chromosome 9. Despite its specific expression in pluripotent cells and germ cells, ESG1 is dispensable for self-renewal of ES cells and establishment of germcells.
Embryonic stem (ES) cells were first derived from the blastocysts of mice in 1981 [1, 2] and humans in 1998 . ES cells have two important properties: theability to maintain pluripotency, which is the ability to differentiate into a wide variety of cells, and rapid proliferation. These characteristics make mouse ES cells an essential component of gene targeting technology. These qualitiesalso make human ES cells attractive sources for cell transplantation therapy to treat various diseases, including spinal cord injuries and juvenile diabetes. The molecular mechanisms underlying the pluripotency and rapid proliferation of ES cells are currently a major focus of the field of stem cell biology [4, 5, 6].
To identify molecules essential in ES cells for these properties, several groups have utilized transcriptome analyses to identify genes specifically expressed in ES cells and early embryos. These analyses, including DNA microarray analyses  and expressed sequence tag analyses [8, 9, 10, 11, 12], identified several common transcripts, including ESG1 that was also designated dppa5 or ECAT2.
ESG1 was originally identified as a transcript Ph34 that was down-regulated by retinoic acid in embryonic carcinoma cells . The expression of this gene was confined in mice to early embryos and germ cells . It is also expressed in pluripotent cells, including ES cells, embryonic germ cells, and multipotent germline stem cells . ESG1 encodes a polypeptide of 118 amino acids that contains a single KH domain, which is an RNA-binding domain . It remains unclear, however, if ESG1 functions as an RNA-binding protein or the roles it plays in ES cells and mice.
Previous genomic library screening by identified genomic clones containing the mouse ESG1 gene and seven pseudogenes . Two of these pseudogenes exhibit a similar exon-intron structure as the ESG1 gene, indicating their generation by gene duplication. The five remaining pseudogenes did not contain any introns, indicating that these were generated by retrotransposition of ESG1 transcripts. The chromosomal localizations of the mouse ESG1 gene and pseudogenes, however, have not been reported.
In this study, we determined the structure of the mouse gene encoding this protein and related pseudogenes. We also performed gene targeting to determine the physiological function of ESG1.
Results and discussion
Chromosomal localization and structures of mouse ESG1gene and psedogenes
On chromosome 9, we identified a DNA fragment similar to the ESG1 gene that included two putative introns. These putative first and second exons, however, contained (4) multiple mutations of the ESG1 cDNA sequence. The putative third exon was identical to that of the previously reported ESG1 gene. Also on chromosome 9, we identified another DNA fragment that was similar, but not identical, to the third exon of the ESG1 gene. These findings suggest that these ESG1-like sequences on chromosome 9 have not been correctly assembled.
We also found that the two BAC clones contained another ESG1-like sequence (Figure 2A). The two sequences, separated by a 68 kbp intergenic sequence, were oriented in opposite directions. The ESG1-like sequence exhibited greater than 95% identity to the exons and introns of the ESG1 gene. This sequence, however, contained critical nucleotide substitutions in all of the exons and one nucleotide insertion in exon 2 (Figure 2B). Although 675 base pairs of the 3' flanking regions were conserved between the ESG1 gene and the pseudogene, only five base pairs of the 5' flanking region were identical. This 5' flanking region (~6 kbp) did not possess any promoter/enhancer activity in luciferase reporter assays (Figure 3). It is thus unlikely that this sequence is transcribed or translated into a functional protein. This sequence likely represents a duplication pseudogene. Bierbaum previously reported the existence of two pseudogenes with similar exon-intron organization as the ESG1 gene . We could not determine which of these two pseudogenes corresponds to the one we identified or the location of the remaining pseudogene.
Targeted disruption of the mouse ESG1gene
To obtain homozygous mutant ES cells, we introduced the β-geo vector into HygR heterozygous ES cells. Of 105 G418-resistant colonies tested, 49 were homozygous for ESG1 deletion. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells (Figure 4C). In 29 clones, the β-geo vector had replaced the HygR vector, such that the cells remained heterozygous. In the remaining 27 clones, the β-geo vector was integrated at non-homologous sites.
To generate ESG1-knockout mice, we injected β-geo-ESG1+/- ES cell clones into the blastocysts of C57BL6 mice. We obtained germline transmission from three clones. We obtained ESG1-/- mice at the Mendelian ratios (36 wild-type, 69 ESG1+/-, and 45 ESG1-/-) from intercrosses of ESG1+/- mice. These animals exhibited normal development, gross appearance, and fertility (not shown). Histological examination of testis and ovary could not identify any abnormalities (not shown). These data demonstrated that ESG1 is dispensable for both mouse development and germ cell formation.
We also generated ES cells from blastocysts obtained by intercrosses of ESG1+/- males and ESG1-/- females. Of the eight ES cell lines established, two clones were ESG1-/-. These ESG1-null ES cells demonstrated normal morphology, proliferation, and differentiation (not shown), confirming that ESG1 is dispensable in ES cells.
To analyze the physiological roles of ESG1, we identified the mouse gene on chromosome 9 and deleted it by homologous recombination in ES cells. Despite specific expression in early embryos, germ cells, and pluripotent cells, our data demonstrated that ESG1 is dispensable for mouse development, germ cell formation, and ES cell self-renewal.
Identification and analyses of BAC clones containing the mouse ESG1gene
To identify bacterial artificial chromosome (BAC) clones containing mouse ESG1 gene, we performed PCR-based screening of mouse BAC library DNA pools (Research Genetics) using the pH34-u38 (5'-GAAGTCTGGTTCCTTGGCAGG-3') and pH34-L394 (5'-ACTCGATACACTGGCCTAGC-3') primers. Following restriction enzyme digestion, we performed Southern blot analyses of BAC clones as described  using the pH34-U258 (5'-CTCGAGTGTACAGTCAAGTGGTTGCTGGGA-3'), pH34-U65 (5'-GTGACCCTCGTGACCCGTAA-3'), pH34-intron1L (5'-CTGCGTGAGAGAAACACCAAACAGGC-3'), pH34-L545 (5'-TGTGAATGGGAAGGTTACCACTCT-3') and pH34-SCL1 (5'-GCCCTCTTCTGGTTTGTCTCGAAAT-3') probes. Hybridization with these probes revealed bands containing either the ESG1 gene or pseudogenes.
To sequence the region containing the mouse ESG1 gene and the 3' flanking region, we subcloned a ~15 kbp XhoI-SalI fragment into the pZERO-2 vector (Invitrogen). HindIII- or EcoRI-digested fragments of this vector were then cloned into pBluescript KS(-) for sequencing. To sequence the ESG1 pseudogene and the 3' flanking region, an 8 kbp NotI/XhoI fragment was cloned into pBluescript KS(-). BamHI- or PstI- fragments of this vector were also cloned into pBluescript KS(-). To identify the sequence containing the 5' flanking regions of the ESG1 gene and the related pseudogenes, we used a TOPO walker kit (Invitrogen) with the pH34-T2L (5'-ACTAGTCGCAGCAGGGATCCAGGAATATCT-3') and pH34-L394 primers. The resulting sequence was cloned into pCR2.1 (Invitrogen). We obtained a ~6 kbp band from the NsiI-digensted library; XbaI-, SpeI-, EcoRI-, and PstI-digested fragments of this band were cloned into pBluescript KS(-) for sequencing. This fragment contained the 5' flanking region of the ESG1 gene. A ~3 kbp fragment, obtained from the SacI-digested library, was cloned into pCR2.1 for sequencing. This fragment was contained the 5' region flanking the pseudogene.
Construction of ESG1targeting vectors
We replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette  or an IRES-HygR cassette by promoter trap selection. We amplified the 5' arm (1.8 kbp) using KOD plus (TOYOBO) with the pH34-targetpair5-U (5'-CCGCGGAAAGTCAAGAGATTGGGTGG-3') and pH34-targetpair5-L (5'-GCGGCCGCCTTTACGGGTCACGAGGGTCAC-3') primers. The 3' arm (5.8 kbp) was amplified using the pH34-targetpair3-U (5'-TGTGGCCAGTGTTTGGTTCTGGCGGG-3') and pH34-targetpair3-L (5'-CTCGAGGACTCGCCATTCTAGCCAAG-3') primers. The IRES β-geo or IRES HygR cassettes were ligated in between the two PCR fragments. The diphtheria toxin A cassette was placed downstream of the 3' arm. After linearization with SacII, these targeting vectors were electroporated into 2.0 × 107 RF8 ES cells  using a Gene pulser (BIORAD). Transfected cells were selected with 250 μg/mL G418 or 100 μg/mL hygromycin B, respectively. Genomic DNA from G418- or hygromycin B-resistant colonies was screened for homologous recombination by Southern blotting.
Southern blot screening for homologous recombination
ES cells genomic DNA was extracted with PUREGENE™ Cell Lysis Solution (Gentra systems). For 5' Southern blot analysis, genomic DNA was first digested with PstI, then separated on an 0.8% agarose gel and transferred to a nylon membrane as described . A 560 bp 5' probe was amplified using the ESG1S5 (5'- GATGGTGGTGGTGACTCAGAG -3') and ESG1AS5 as (5'- CCTCCATTGCCTCTATATCAG -3') primers. The probe specifically labeled an 18 kbp band from the wild-type locus, a 15 kbp band from the β-geo locus, and a 12 kbp band from the HygR locus. Genomic DNA was also digested with SpeI for 3' Southern blot analysis. A 1,010 bp 3' probe was amplified with the pH34U-8000 (5'- CCAACCAGCCAGAGTTTCAGTTAT -3') and pH34L-9000 (5'-GATAAGCTGCTGCCAAAAGACAAG -3') primers. The probe hybridized to an 11.5 kbp band from the wild-type locus, a 12.5 kbp band from the β-geo locus, and a 9.5 kbp band from the HygR locus.
Generation of anti-ESG1 polyclonal antibodies
The coding sequence of Esg1 was amplified by PCR with the pH34-gw-s (5'- AAAAAGCAGGCTGGATGATGGTGACCCTCGTGA-3') and pH34-gw-as (5'- AGAAAGCTGGGTCTGCATCCAGGTCGGAGACA-3') primers. To construct pDONR-pH34, the resulting PCR product was subcloned into pDONR201 (Invitrogen). pDONR-pH34 was interacted with pDEST17 (Invitrogen) by LR recombination. After introduction of the resulting expression vector pDEST17-pH34 into BL21-AI E. coli (Invitrogen), recombinant protein production was induced according to the manufacture's protocol. Histidine-tagged ESG1 was purified using Ni-nitrilotriacetic acid agarose (Qiagen) under denaturing conditions in the presence of 8 M urea. After dialysis against 6 M urea, the recombinant proteins were injected into New Zealand White rabbits to generate anti-ESG1 polyclonal antibodies.
After preparation of ES cell extracts with M-Per (Pierce), cellular proteins were separated on sodium dodecyl sulfate (SDS)-14% polyacrylamide gels and transferred to nitrocellulose membranes (Millipore). Membranes were incubated with anti-ESG1 (1/500 dilution), anti-Oct3/4 (1/500; Santa Cruz Biotechnology), anti-CDK4 (1/200; Santa Cruz Biotechnology), and anti-GFP (1/1000; MBL) primary antibodies. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (1/5000; Cell Signaling) were used to detect antibody binding. We visualized bound antibody with an ECL Western Blotting Detection System (Amersham).
Derivation of ESG1-deficient ES cells from blastocysts
Esg1+/-or ESG1-/- mutant female mice were injected with Tamoxifen (10 μg) and Depo-provera (1 mg) subcutaneously on the third day of pregnancy. Four days later, embryos in diapause were flushed out of the uterus and cultured on STO feeder cells in four-well plates in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% Fetal Bovine Serum (Hyclone), 0.1 mM Non-Essential Amino Acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 U/ml Penicillin-Streptomysin (Invitrogen), and 0.11 mM 2-mercaptoethanol (Invitrogen). After six days, the central mass of each explant was harvested, rinsed in PBS, and placed in a drop of trypsin for a few minutes. The cell mass was collected with a finely drawn-out Pasteur pipette preloaded with medium, ensuring minimal carryover of the trypsin. The cells were gently transfered into a fresh well with 20% FBS-containing medium. The resulting primary ES cell colonies were individually passaged into wells of four-well plates containing STO feeder cell layers. Thereafter, cells were expanded by trypsinization of the entire culture.
Total RNA from wild-type ES cells and ESG1-/- ES cells was labeled with Cy3 and Cy5, respectively. The samples were hybridized to a Mouse Development Microarray (Algilent) according to the manufacturer's protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent). Hybridization was repeated with two independent clones. Data were analyzed with GeneSprings software (Silico Genetics).
We thank Chihiro Takigawa, Junko Iida, Masako Shirasaka, Yumi Ohuchi, and Megumi Narita for their technical and administrative assistance. We are grateful to Drs. Minoru Ko and Azim Surani for sharing helpful data prior to publication, Dr. Hitoshi Niwa for generously providing various vectors, and Dr. Robert Farese Jr. for his kind gift of the RF8 ES cells. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Uehara Memorial Foundation, the Naito Foundation, the Sumitomo Research Foundation, the Mitsubishi Foundation. S.Y. was supported by a Toray Science and Technology Grant (to S.Y.). This work was also supported in part by a Grant-in-Aid for 21st Century COE (Center of Excellence) Research from the Ministry of Education, Culture, Sports, Science, and Technology.
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