AB2.2細胞

品系： 129/SvEvBrd- Hprt

器官來源： 胚胎

組織來源： Inner cell mass

細胞類型： 胚胎干細胞

是否是腫瘤細胞： 0

物種來源： 小鼠

數量： 大量

年限： Embryo, blastocyst

ATCC Number： SCRC-1023?

細胞形態： 其他

運輸方式： 凍存運輸

AB2.2細胞Designations: AB2.2

Depositors: A Bradley

Biosafety Level: 1

Shipped: frozen

Medium & Serum: See Propagation

Organism: Mus musculus

Morphology: Spherical colony

Source: Organ: embryo

Cell Type: embryonic stem cell;

Strain: 129/SvEvBrd- Hprtb-m2

Tissue: Inner cell mass

Permits/Forms: In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. AB2.2細胞Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location.

Restrictions: Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact Baylor Licensing Group, blg@bcm.tmc.edu, 713-798-6821.

Isolation: Isolation date: 1988

Applications: Differentiation: AB2.2 cells have been successfully differentiated into cardiomyocytes from embryoid bodies.

Gene knock-out: AB2.2 retain very high germline transmission rates after being genetically manipulated making them an excellent candidate for targeted-mutation and gene knock-out experimentation. They have been used to create knock-out mice to study the role of epidermal proteins in normal development.

Gene knock-down: AB2.2 cells have been used in RNA interference research aimed at understanding the role of various proteins in tissue-specific development and function

Recombineering: A unique, fully end-sequenced, 129Sv BAC library consisting of 84,507 bacterial artificial chromosomes has been generated from AB2.2 ES cell DNA. This BAC library, referred to as bMQ BAC (www.geneservice.co.uk), is a publicly available BAC resource that can be used for the rapid construction of targeting vectors using current recombineering techniques.

AB2.2細胞Age: Embryo, blastocyst

Gender: male

Comments: The line has been used extensively for creation of knockout mice. The HPRT negative mutation makes the line useful for chromosome engineering. This mouse ES cell line has been shown to be germline competent.

Propagation: ATCC complete growth medium: Mouse ES Cell Basal Medium

Temperature: 37.0°C

Growth condition: feeder cells required

Subculturing: Protocol: Establishing and maintaining your culture: To insure the highest level of viability, be sure to warm media to 37C before using it on the cells.

Plate mitotically arrested MEF (CF-1) (ATCC SCRC-1040)as a feeder layer. Refer to the product sheet for mitotically arrested MEF for detailed handling instructions. One hour before thawing the vial of ES cells, perform a 100% medium change using complete growth medium for ES cells.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 90 seconds).

Remove the vial from the water bath before the contents are completely thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

Transfer the vial contents plus 5 mL of complete growth medium for ES cells to a 15 mL centrifuge tube. Use an additional 1 mL of media to rinse the vial and transfer the liquid to the 15 mL tube. Add 4 mL of complete growth medium for ES cells to bring the total volume to 10 mL.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant and resuspend the pellet in 2 mL of complete growth medium for ES cells.

Add the 2 mL of cell suspension to the appropriate size flask containing feeder cells and fresh complete growth medium (see batch specific information). AB2.2細胞ES cells should be plated at a density of 30,000 to 50,000 cells/ cm2.

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 to 2 days.

Subculturing Procedure: To insure the highest level of viability, be sure to warm media and Trypsin - EDTA to 37°C before using it on the 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 every 1 to 2 days is recommended. Plating densities should range from 30,000 - 50,000 cells/ cm2. Note: If the colonies are close to or touching each other the culture is overgrown . Overgrowth will result in differentiation.

Prepare enough flasks with MEFs as stated above in step #1.

Aspirate the medium from the flask(s) with the ES cells.

Wash with PBS (Ca+2/Mg+2-free, ATCC SCRR-2201).

Add 3.0 ml of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution (ATCC 30-2101) to each T75 and place the flasks in the incubator. After one minute the ES colonies will dissociate and the cells will begin to detach from the flask.

Dislodge the cells by gently tapping the side of the flask then wash the cells off with 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.

Transfer the cell suspension to the appropriate size centrifuge tube.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant.

Resuspend in 30 to 50 mL of fresh culture medium, depending on the split ratio.

Aspirate the medium from flasks containing feeders and replace it with cell suspension (15 mL/flask).

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 to 2 days.

Medium Renewal: Every day.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:7 is recommended.

Preservation: Storage temperature: liquid nitrogen vapor phase

Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.

References: 16173101: Bader A, Al-Dubai H, Weitzer G. Leukemia Inhibitory Factor Modulates Cardiogenesis in Embryoid Bodies in Opposite Fashions. Circ. Res. 86: 787-794, 2000. PubMed: 10764413

16173102: Wolcik SM, Longley MA, Roop DR. Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b. J. Cell Biol. 154: 619-630, 2001. PubMed: 11489919

16173103: Liu Y, et al. Sox17 is essential for the specification of cardiac mesoderm in embryonic stem cells. Proc Natl Acad Sci USA. 104(10): 3859-3864, 2007. PubMed: 17360443

16173104: Adams DJ, et al. A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics 86(6): 753-758. 2005. [PubMed: 16257172]

16173130: Festing MF, et al. Revised nomenclature for strain 129 mice. Mamm. Genome. 10(8):836, 1999.[PubMed: 10430671]

16173166: Simpson EM, et al. Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat. Genet. 16(1):19-27, 1997. PubMed: 9140391

16173413: Bradley A, Zheng B, Liu P. Thirteen years of manipulating the mouse genome: a personal history. Int. J. Dev. Biol. 42 (7): 943-950, 1998. PubMed: 9853825