R1, a mouse embryonic stem cell line
Two innovations are associated with R1 ES cells:
1. The development of a simplified method of introducing the ES cell genome into mice by generating germline transmitter chimeras using aggregationof a clump of ES cells and 8-cell stage embryo into a microdepression made in the bottom of the tissue culture dish.
This made the technology available to laboratories equipped with ordinary tissue culture and having access to an animal facility. There is no longer a need for expensive equipment and specially acquired technical skills such as the efficient operation of micromanipulators.
2. The development of a technology to produce completely ES cell-derived embryos by combining ES cells with tetraploid embryo. The tetraploid compartment develops into the placenta and some extraembryonic membranes and is selected against in the embryo proper, leaving this lineage completely ES cell-derived.
Material Transfer Agreement - Please send request to Sandy Martino <martino@mshri.on.ca>
What to do with the vial of R1 you have received
Distinguishing features of R1:(back)
1. Established in August 1991, from (129X1/SvJ x 129S1)F1 3.5-day blastocyst. The cells are heterozygous for the c locus (+/c(ch)) and for the pink eye locuc (+/p). The c(ch) and the p alleles are from the 129X1/SvJ parent. In the F1 generation the coat colour is uniform agouti, while in the F2 these two coat colour 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.
2. Sex: male.
3. Pluripotency of R1 was initially tested by tetraploid embryo <-> ES aggregates for completely ES derived development(Nagy et al., 1993) and by diploid embryo <-> ES aggregates and blastocyst injection for germline transmission in chimeras(Wood et al., 1993).
4. 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 we tested by tetraploid aggregates(Nagy et al., 1993).
5. R1-derived animals reached adulthood and were fertile.
6. 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.
7. More than 90% of the individual K.O. clones went to germline (n>60) by aggregation chimeras in our hands.
8. For aggregation chimeras, specially designed darning needles are manufactured by:
Biologcal Laboratory Equipments and Services Ltd.
H-1165 Budapest, Zselyi Aladar u.31
Hungary
Tel/Fax: (361) 407-2602
They also manufacture an instrument for embryo fusion and different light sources for GFP visualization.
Frozen stock:
For general distribution, we froze down a large number of vials of passage #10 and #11. This stock was tested at the time of freezing with tetraploid aggregation chimeras and gave good developmental rate to term (9 of 48 aggregates transferred) and recovery after birth (3 of 9 newborns).
The pool was tested negative for mycoplasma.
What to do with the vial of R1 you have received:(back)
To create an early passage pool of R1:
ES medium
DMEM high glucose
+ 0.1 mM non-essential amino acids (100x stock from GIBCO)
+ 1 mM sodium pyruvate (100x stock from GIBCO)
+ 100 microM beta-mercaptoethanol (from 10-2 M stock (100x), Sigma)
+ 15% fetal bovine serum
+ penicillin and streptomycin (final concentration 50 microgram/ml each)
+ 1000-2000 U LIF/ml
1. Thaw the vial quickly and transfer its contents slowly into a centrifuge tube containing 5 ml of ES medium.
2. Spin down and resuspend the pellet in 5 ml of ES medium and plate to 60 mm tissue culture plate with primary embryonic feeders.
3. Change the medium the next day (every day).
4. The cells are usually ready two days after thawing for passing into 1 or 2 x 100 mm plates with feeders.
5. Pass the cells 1:5 to 1:7 if the confluency reaches 20-40% of surface area. Usually you have to do this every other day.
6. If you have enough large plates, freeze four vials from each plate to create your own early passage pool.
For details regarding the culture conditions, electroporation, selection, picking, screening, etc., consult (Nagy et al. 2002, Wurst and Joyner, 1993; Nagy and Rossant, 1993, 1999; Nagy, 1997; Pirity et al., 1998)
Maintenance and conditions: (back)
1. We prefer to keep the cells on feeders, although we have had germline transmission from experiments done on gelatinized plates also. They just look better on feeders.
2. Originally R1 was established on STO feeders, but later we found that primary embryonic fibroblast feeders are better for the cells than the STO line that we had.
3. We use mitomycin C to inactivate feeders just before plating them on the final tissue culture plates.
4. We also like to supplement the medium with LIF (1000 U).
5. Some Fetal Bovine S erum (FBS), even if they work fine for other ES cell lines, differentiate R1. Several FBS sources should be tested for maitaining undifferentiated stage and high chimeric contribution.
References:(back)
Nagy A., Gertsensten M., Vintersten K. and Behringer R. (2002) Manipulating the Mouse Embryo; A Laboratory manual. 3rd edition, Cold Spring Harbor Press , Cold Spring Harbor, New York
Nagy, A. and J. Rossant (1999). Production and analysis of ES-cell aggregation chimeras. In Gene Targeting: A Practical Approach (ed. A. Joyner, Second Edition) , Oxford University Press Inc., New York, pp. 177-206.
Nagy, A. and Rossant, J. (1993). Production of completely ES cell-derived fetuses. In Gene Targeting: A Practical Approach. (Ed. A. Joyner), 147-179. IRL Press at Oxford University Press,
Nagy, A. (1997). Formation of mouse chimeric embryos from ES cells. In Transgenic Animals: Generation and Use. (Ed. L. M. Houdebine), 167-172. Harwood Academic Publishers, Amsterdam.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. (1993). "Derivation of completely cell culture-derived mice from early-passage embryonic stem cells." Proc. Natl. Acad. Sci. USA 90, 8424-8428.
Pirity, M., Hadjantonakis, A.-K. and Nagy, A. (1998). Embryonal stem cells, creating transgenic animals. In Cell Culture for Cell and Molecular Biologists. in press. (Ed. J. P. Mather and V. Barnes), Academic Press, San Diego.
Wood, S. A., Allen, N. D., Rossant, J., Auerbach, A. and Nagy, A. (1993). Non-injection methods for the production of embryonic stem cell-embryo chimeras. Nature, , 365, 87-89.
Wurst, W. and Joyner, A. (1993). Production of targeted embryonic stem cell clones. In Gene Targeting: A Practical Approach. (Ed. A. Joyner), 33-62. IRL Press at Oxford University Press,