Validation of new mES cell lines

5.1 Karyotype

The first stage in validating a new mES cell line is to determine its sex and ensure that it has a normal karyotype. Much time and effort can be wasted in pursuing work with a line that is subsequently shown to be karyotypically abnormal. Loss of an X chromosome, and trisomies 8 and 11, are common abnormalities. Hence it is vital to analyse the exact chromosome constitution; a simple count cannot, for instance, distinguish between a normal karyotype and one that is XO with trisomy 8. We discard any line in which more than 10% of cells are trisomic, as in our experience the proportion of such cells may increase rapidly with successive passages.

5.2 Pluripotency

To demonstrate unequivocally that a newly derived mES cell line is pluripotent requires the generation of chimeras among whose offspring are individuals sharing the cell line's genetic background: that is, the mES cell line is germline competent. There are two principal methods for producing chimeras using mES cells, by blastocyst injection or morula aggregation, for which various protocols are available (24, 25, 31, 32). To be useful for genetic modification, a novel mES

Figure 5 Stages of blastocyst dissection (modified with the authors' and publisher's permission from (28)). All views are as seen through the microscope, looking vertically down onto the blastocyst through the coverslip. (A) The blastocyst is stabbed with the needles, away from the ICM, and raised until it is in contact with the coverslip. The blastocyst is positioned with the ICM to one side, away from where the tear will be made. (B) The two needles are moved apart, as indicated by the arrow, to tear the trophectoderm open. The blastocyst is then incubated in trypsin/pancreatin solution. (C) Once returned to the chamber drop, one needle is pushed into the trophectoderm and the blastocyst is raised up against the coverslip. The second needle is inserted and the two moved apart, to spread the trophectoderm against the coverslip as a horizontal sheet of cells. (D) One needle is left in position holding the blastocyst whilst the second is moved to the same side of the ICM, lowered slightly, and used to scrape away the endoderm. (E) Once this manoeuvre is started, the endoderm should peel away readily. (F) The second needle should be returned to the starting position as in (D), raised slightly, and used to scrape away the epiblast from the overlying polar trophectoderm.

Figure 5 Stages of blastocyst dissection (modified with the authors' and publisher's permission from (28)). All views are as seen through the microscope, looking vertically down onto the blastocyst through the coverslip. (A) The blastocyst is stabbed with the needles, away from the ICM, and raised until it is in contact with the coverslip. The blastocyst is positioned with the ICM to one side, away from where the tear will be made. (B) The two needles are moved apart, as indicated by the arrow, to tear the trophectoderm open. The blastocyst is then incubated in trypsin/pancreatin solution. (C) Once returned to the chamber drop, one needle is pushed into the trophectoderm and the blastocyst is raised up against the coverslip. The second needle is inserted and the two moved apart, to spread the trophectoderm against the coverslip as a horizontal sheet of cells. (D) One needle is left in position holding the blastocyst whilst the second is moved to the same side of the ICM, lowered slightly, and used to scrape away the endoderm. (E) Once this manoeuvre is started, the endoderm should peel away readily. (F) The second needle should be returned to the starting position as in (D), raised slightly, and used to scrape away the epiblast from the overlying polar trophectoderm.

cell line should give chimeras at a rate of at least 50% of the total number of pups born, and most of the male chimeras should transmit through the germline. For purposes that do not require the generation of chimeric mice per se, such as in vitro differentiation experiments, a lower rate of chimera production may be acceptable.

Mouse ES cells are characterized by markers including the stage-specific embryonic antigen, SSEA-1 (33), alkaline phosphatase activity, and expression of the genes Oct4 (34-36) and Nanog (37-38). Expression of these markers is consistent with - but not evidence of - pluripotency. However, if the pluripo-tency of a novel cell line is not an issue, it may be sufficient to use the presence of such markers to demonstrate mES cell-like characteristics.

Figure 6 Stages in the derivation of mES cells from cultured eplblasts. (A) Epiblast Isolated from day 4.5 p.c. blastocyst and cultured on feeder cells for 3d. The colony is small with clearly defined boundaries, and individual cells are not distinguishable. (B) Contaminating colony of extraembryonic endoderm cells in epiblast culture. The individual cells are clearly distinguishable, with highly refractile cell borders. (C) Epiblast isolated from delayed-implanting blastocyst and cultured on feeder cells for 5d. The colony has a clearly defined boundary and resembles in morphology a single, large mES-cell colony. (D) Cultured mES-cell colonies on feeder cells, 4d after the second dissociation of isolated epiblast. Scale bars: 100 mm.

Figure 6 Stages in the derivation of mES cells from cultured eplblasts. (A) Epiblast Isolated from day 4.5 p.c. blastocyst and cultured on feeder cells for 3d. The colony is small with clearly defined boundaries, and individual cells are not distinguishable. (B) Contaminating colony of extraembryonic endoderm cells in epiblast culture. The individual cells are clearly distinguishable, with highly refractile cell borders. (C) Epiblast isolated from delayed-implanting blastocyst and cultured on feeder cells for 5d. The colony has a clearly defined boundary and resembles in morphology a single, large mES-cell colony. (D) Cultured mES-cell colonies on feeder cells, 4d after the second dissociation of isolated epiblast. Scale bars: 100 mm.

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