Sox9 and Oct4 are two important regulatory factors involved in mammalian

Sox9 and Oct4 are two important regulatory factors involved in mammalian development. of the tissue- and site-specific expression of these proteins. The dynamics of the methylation of 15 CpGs and six CpGs in Sox9 and Oct4 respectively was studied with sodium bisulfite genomic DNA sequencing in mouse testis at different developmental stages. Consistent methylation of three CpGs was observed in adult ovary in which the expression of Sox9 was feeble while the level of methylation in somatic tissue was greater in Oct4 compared to germinal tissue. The promoter-chromatin status of Sox9 was also studied with a chromatin immune-precipitation assay. methylation and expression pattern in the promoter region of mouse Sox9 and Oct4 gene during development. The correlation between the expression of these genes in the different developmental IC-87114 stages of testis and somatic tissues and methylation was also examined. Fifteen sites for Sox9 gene and six sites for Oct4 gene was investigated in somatic and germinal tissues of different developmental stages. Materials and Methods Genomic DNA extraction Parkes strain of mice of both sexes was used in this study and the experimental protocols were approved by the institutional ethics committee. The mice were housed in groups of 6-7 per polypropylene cage (43 × 27 × 15 cm) under standard laboratory conditions. Mating was achieved by housing one male mouse with two female mice in separate cages. Pregnant females were isolated and housed separately after the detection of vaginal plug the next morning. When required the mice were killed by cervical dislocation and selected germinal and somatic tissues were excised blotted free of blood and weighed. DNA was isolated from embryonic neonatal and adult testes. Kidney and ovary were used as a source of somatic tissue DNA. Genomic DNA from adult and neonatal stages was isolated from three independent mice. Multiple MGCs (mesonephron IC-87114 gonadal complexes) were pooled from different embryos of the same stage e.g. 11.5 dpc and 18.5 dpc to minimize the chance of contamination from other tissues. DNA was isolated with the help of standard protocol using Proteinase K digestion (50 μg/mL) at 37°C for 12-14 h. Phenol: chloroform: isoamylalcohol (25:24:1) extraction was done at 25°C. Finally DNA was precipitated with 1/30 volume of 3 M sodium acetate (pH 5.0) and two volumes of chilled absolute ethanol. Sodium bisulfite treatment The Bisulfite conversion was carried out according to the protocol of Clark (1994) with IC-87114 minor modifications. Briefly 1 μg DNA in a volume of 50 μL was denatured by adding 3 μL of 5 M NaOH and incubating IC-87114 for 15 minutes at 37°C. After denaturation 420 μL of 3.9 M (saturated) sodium bisulfite (Sigma final concentration 3.4 M; pH 5.0) and 33 μL of 20 mM hydroquinone (Sigma final concentration 0.58 mM) were added to the denatured DNA and incubated at 50°C for 12-14 h. Treated DNA was desalted and purified using the Wizard DNA Clean-Up system (Promega USA) desulfonated by adding 3 μL of 5 M freshly Rabbit Polyclonal to SIX3. prepared NaOH followed by incubation for 15 minutes at 37°C and finally precipitated with 1 μL of glycogen (Fermentas final concentration 150 μg/mL 25 μL of 10 M ammonium acetate (pH 7.0) and 150 μL of chilled absolute ethanol. The precipitated DNA was pelleted and resuspended in 40 μL of sterile water and stored at -20 °C until used. PCR of bisulfite-treated DNA Approximately 100-150 ng of bisulfite-treated DNA was used for each PCR. HotStar polymerse (Qiagen) was used for amplification of bisulfite-converted DNA. PCR conditions were 94°C for 4 min followed by 94°C for 1 min 60 for 1 min for Sox9 and 58°C for Oct4 and 72°C for 1 min for 35 cycles with a final extension at 72°C for 6 IC-87114 min. The primer pairs were used to amplify the 250 bp region (from base pairs 1 to 250) for Sox9 and the 145 bp region (from base IC-87114 pairs 2641 to 2786) for Oct4 gene. The primers sequences of Sox9 are FP: 5-GTTGTGGAGGG TTTTAGTTTAGATA-3 and RP 5′-AAAAAAAACTC AACCAAAAAA TAAATAATA-3; Oct4 gene FP: 5 RP 5′-CCACC CTCTAACCTTAACTCCTAAC-3′. The GenBank accession numbers for Sox9 and Oct4 gene are “type”:”entrez-nucleotide” attrs :”text”:”AB022193.1″ term_id :”4587259″ term_text :”AB022193.1″AB022193.1 and {“type”:”entrez-nucleotide” attrs :{“text”:”AJ297528.1″ term_id :”11602787″.