PMC:1420271 / 14426-21475 JSONTXT

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and analyses of BAC clones containing the mouse ESG1 gene\nTo 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 [20] 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.\nTo 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.\n\nConstruction of ESG1 targeting vectors\nWe replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette [21] 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 [22] 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.\n\nSouthern blot screening for homologous recombination\nES 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 [20]. 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.\n\nGeneration of anti-ESG1 polyclonal antibodies\nThe 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.\n\nWestern blot\nAfter 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).\n\nDerivation of ESG1-deficient ES cells from blastocysts\nEsg1+/-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.\n\nMicroarrays\nTotal 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)."}

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and analyses of BAC clones containing the mouse ESG1 gene\nTo 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 [20] 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.\nTo 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.\n\nConstruction of ESG1 targeting vectors\nWe replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette [21] 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 [22] 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.\n\nSouthern blot screening for homologous recombination\nES 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 [20]. 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.\n\nGeneration of anti-ESG1 polyclonal antibodies\nThe 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.\n\nWestern blot\nAfter 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).\n\nDerivation of ESG1-deficient ES cells from blastocysts\nEsg1+/-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.\n\nMicroarrays\nTotal 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)."}

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,"obj":"T11960"},{"id":"R3460","pred":"punct","subj":"T11962","obj":"T11960"},{"id":"R3461","pred":"punct","subj":"T11963","obj":"T11954"}],"text":"Methods\n\nIdentification and analyses of BAC clones containing the mouse ESG1 gene\nTo 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 [20] 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.\nTo 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.\n\nConstruction of ESG1 targeting vectors\nWe replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette [21] 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 [22] 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.\n\nSouthern blot screening for homologous recombination\nES 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 [20]. 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.\n\nGeneration of anti-ESG1 polyclonal antibodies\nThe 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.\n\nWestern blot\nAfter 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).\n\nDerivation of ESG1-deficient ES cells from blastocysts\nEsg1+/-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.\n\nMicroarrays\nTotal 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)."}