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Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office (2007). The entire text is available online at

http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

• Biomarkers: Included in microarray studies aimed at developing methods for detecting novel marker ES genes [PubMed: 17875203], as well as biomarker signature patterns expressed during differentiation [PubMed: 17605829]. 
• Embryonic development: Used to study X chromosome inactivation [PubMed: 11713286 and PubMed:9618446] and DNA methylation involved in gene regulation [PubMed: 17182866,PubMed:12370304, PubMed: 8917520 and PubMed: 11884600]. 
• Cancer Research: Cre-Lox generation of a conditional VHL null allele in J1 cells has been used to generate Chimeric mice for the study von Hippel-Lindau (VHL) syndrome [PubMed: 11171994].

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
2. One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Dissociation and Transfer of ES Cells

1. Aspirate the medium from the flask(s) containing ES cells.
2. Wash with PBS Ca+2/Mg+2-free (ATCC 30-2200).
3. Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
4. Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
6. Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm².
7. Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
8. Incubate the culture at 37°C in a humidified 5% CO₂/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.

Stevens LC. A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Natl. Cancer Inst. 50: 235-242, 1973. PubMed: 4692863

Li E, et al. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915-926, 1992. PubMed: 1606615

Krzyzanowski PM, et al. Identification of novel stem cell markers using gap analysis of gene expression data. Genome Biology 8(9): R193, 2007. PubMed: 17875203

Luikenhuis S, et. al. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells. Mol. Cell Biol. 21(24): 8512?8520, 2001. PubMed: 11713286

Goto T, et al. Regulation of X-Chromosome inactivation in development in mice and humans. Micro. Molec. Biol. Rev. (ASM) 62(2): 362-378, 1998. PubMed: 9618446

Yap DYL, et al. Using biomarker signature patterns for an mRNA molecular diagnostic of mouse embryonic stem cell differentiation state. BMC Genomics 8: 210, 2007. PubMed: 17605829

Haase VH, et al. Vascular tumors in livers with targeted inactivation of the von Hippel?Lindau tumor suppressor. Proc. Natl. Acad. Sci. 98(4): 1583?1588, 2001. PubMed: 11171994

Oda M, et al. DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner. Genes Dev. 20(24): 3382?3394, 2006. PubMed: 17182866

Lorincz MC, et al. DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol. Cell Biol. 22(21): 7572?7580, 2002.PubMed: 12370304

Tucker KL, et al. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25: 3745-3746, 1997. PubMed: 9278500

Biniszkiewicz D, et al. Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Mol. Cell Biol. 22(7): 2124?2135, 2002. PubMed: 11884600