R1/E細胞

品系： 129X1 x 129S1

器官來源： 胚胎

組織來源： inner cell mass

數量： 大量

細胞形態： 球形

細胞類型： 胚胎干細胞

是否是腫瘤細胞： 0

物種來源： 小鼠

生長狀態： 貼壁生長

運輸方式： 凍存運輸

ATCC Number： SCRC-1036?

年限： 3.5 days embryo, blastocyst

Designations: R1/E

R1/E細胞Depositors: A Nagy

Biosafety Level: 1

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Mus musculus

Morphology: spherical colony

Source: Strain: 129X1 x 129S1

Organ: embryo

Tissue: inner cell mass

Cell Type: embryonic stem cell;

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Applications: R1/E細胞The segregation could result in several coat types, from albino, through light brown, to black, depending on the genetic background of the partner of the germline chimaera.

The R1 cell line was established in August 1991, from a 129X1 x 129S1 3.5 day blastocyst.

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 [PubMed: 8378314].

Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development [PubMed: 8378314].

Age: 3.5 days embryo, blastocyst

Gender: male

Comments: The R1/E cell line was subcloned from R1 in EMBL, Heidelberg, Germany by Kristina Vintersten. The R1 cell line was established in August 1991, from a 129X1 x 129S1 3.5 day blastocyst. The cells are heterozygous for the c locus (+/c (ch)) and for the pink eye locus (+/p). This mouse ES cell line has been shown to be germline competent.In the F1 generation the coat color is uniform agouti, while in the F2 these two coat color genes segregate. The segregation could result in several coat types, from albino, through light brown, to black, depending on the genetic background of the partner of the germline chimaera.

Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development [PubMed: 8378314]. They were also tested by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras [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 [PubMed: 8378314]. R1-derived animals reached *****hood 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.

Propagation: R1/E細胞ATCC complete growth medium: ES-DMEM (ATCC SCRR-2010) supplemented with 2.0 mM L-Alanyl-L-Glutamine (ATCC 30-2115), 0.1 mM non-essential Amino Acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Invitrogen Life Technologies No. 21985), 1000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon No. ESG1107) and 15% fetal bovine serum (ATCC SCRR-30-2020).

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37.0°C

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

Plate mitotically arrested MEF (CF-1) (ATCC SCRC-1040)as a feeder layer at approximately 5.0 to 6.0 X 10(6) cells/T75 at least one day before plating R1/E cells (see product sheet for mitotically arrested MEF for protocol). One hour before thawing the vial of R1/E ES cells, perform a 100% medium change using 10 ml of complete ES-DMEM (see below for recipe).

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 as soon as the contents are 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?s contents plus 5 ml of complete ES-DMEM 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 ES-DMEM to bring the total volume to 10 ml.

Spin the cells at 270 xg for 5 min. Aspirate the supernatant and resuspend the pellet in 5 ml of complete ES-DMEM.

Add the 5 ml of cell suspension to the T75 flask containing feeder cells and 10 ml complete ES-DMEM.

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 3 to 4 X 10(6) cells/ T75. 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 R1/E ES cells.

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

R1/E細胞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 xg 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 4 to 7 feeder layer flasks and replace it with 15 ml/flask of R1/E cell suspension.

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.

Interval: Every one to two days

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

Medium Renewal: Every day

Preservation: Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO (ATCC 4-X).

Storage temperature: liquid niitrogen vapor phase

Related Products: parental cell line:ATCC SCRC-1011

References: 57459: Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999

71506: 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

71870: Wood SA, et al. R1/E細胞Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature 365: 87-89, 1993. PubMed: 8361547

71871: Nagy A, Rossant JProduction and analysis of ES-cell aggregation chimerasIn: Nagy A, Rossant JGene Targeting: A Practical ApproachOxfordOxford University Press177-206, 1999