| Biosafety Level |
1 [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.]
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
| Comments |
Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras (Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547). At early passages (up to passage #14), one third of the completely R1-derived newborns generated by tetraploid embryo <-> R1 aggregates survived. No live offspring were produced from cells older than passage #14.*
However, about 20% of subclones derived from passage #14 had the original developmental potential of R1 when tested by tetraploid aggregates (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). R1-derived animals reached adulthood and were fertile. The genetically altered lines derived from R1 gave high efficiency of germline transmission either by injecting them into C57 blastocyst or aggregating them with CD-1 or ICR outbred 8-cell stage embryos. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras.
*Current ATCC stocks of R1 cells are beyond passage 14. Current stocks of alternative subclone of R1 cells, designated R1/E (ATCC SCRC-1036), are below passage 14 and have been shown to be germline competent.
|
| Subculturing |
Subculturing Procedure
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
- Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
- 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
- Aspirate the medium from the flask(s) containing 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) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
- 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.
- Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
- 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.
- 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.
- 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.
|
| Passage History |
Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras (Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547). At early passages (up to passage #14), one third of the completely R1-derived newborns generated by tetraploid embryo <-> R1 aggregates survived. No live offspring were produced from cells older than passage #14.* .
However, about 20% of subclones derived from passage #14 had the original developmental potential of R1 when tested by tetraploid aggregates (Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314). R1-derived animals reached adulthood and were fertile. The genetically altered lines derived from R1 gave high efficiency of germline transmission either by injecting them into C57 blastocyst or aggregating them with CD-1 or ICR outbred 8-cell stage embryos. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras.
*Current ATCC stocks of R1 cells are beyond passage 14. Current stocks of alternative subclone of R1 cells, designated R1/E (ATCC SCRC-1036), are below passage 14 and have been shown to be germline competent. |
| References |
Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999
Nagy A, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA : 8424-8428, 1993. PubMed: 8378314
Wood SA, et al. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547
Nagy A, Rossant JProduction and analysis of ES-cell aggregation chimerasIn: Nagy A, Rossant JGene Targeting: A Practical ApproachOxfordOxford University Press177-206, 1999
Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
|
