Deoxyuridine BrdU Cat No 858811 Sigma

• TG-selection medium: mES-cell medium supplemented with 10 mg/ml 6-thiogua-nine (6TG; Cat. No. A-4660, Sigma)

• CAP-selection medium: mES-cell medium supplemented with 50 mg/ml chloramphenicol (Cat. No. C-7795, Sigma)

1 Plate fused mES cybrids onto fresh feeder layers. If selecting against HPRT " or TK" donor cells, culture the cybrids in HAT medium. Include supplementation with pyruvate and uridine for the first 24 h postfusion only.

2 Individual mES-cell colonies should become visible by day 5 after fusion, and can be picked for further expansion and analysis between days 7 and 9.b aIn older publications, HAT medium was supplemented with 10 mM glycine as conversion of serine to glycine involves DHFR, which is inhibited by aminopterin. However, as FCS is extremely rich in glycine, this supplementation is unnecessary.

bAs mES cells divide rapidly it is important to monitor growth on a daily basis, and to pick colonies for expansion before they become too large and differentiated. The morphology of female mES cybrid colonies of different sizes is shown in Figure 5. Note the differentiation that occurs at the periphery of colonies with over-growth (Figure 5C).

Figure 5 Morphology of mES cybrids. (A) Morphology of SNL76/7 feeder cells lweek after plating. (B) A large and clonal mES cybrid colony. Note that morphology remains typical of mES cells, including the multilayered, high density of cells in the cybrid colony, and the outgrowth of cells at the periphery (arrowhead and arrow, respectively). (C) Spontaneous differentiation of mES cybrids, at the periphery of an overgrown colony, into primitive endoderm cells (arrow) with concomitant deposition of Reichert's membrane-like ECM (arrowhead). (D) An optimally sized mES cybrid colony for subcloning. Note the lack of differentiation around the periphery of this colony. Magnifications (A-C) 100 x ; (D) 50 x .

Figure 5 Morphology of mES cybrids. (A) Morphology of SNL76/7 feeder cells lweek after plating. (B) A large and clonal mES cybrid colony. Note that morphology remains typical of mES cells, including the multilayered, high density of cells in the cybrid colony, and the outgrowth of cells at the periphery (arrowhead and arrow, respectively). (C) Spontaneous differentiation of mES cybrids, at the periphery of an overgrown colony, into primitive endoderm cells (arrow) with concomitant deposition of Reichert's membrane-like ECM (arrowhead). (D) An optimally sized mES cybrid colony for subcloning. Note the lack of differentiation around the periphery of this colony. Magnifications (A-C) 100 x ; (D) 50 x .

2.9 PCR-based allele genotyping of the nuclear genome of cybrids

When generating mouse cybrid lines, it is useful to be able to discriminate between the nuclear genomes (nDNA) of the two cell types used to construct the cybrids. This is especially important when drugs or other compounds cannot be used in the culture medium to select for cybrids postfusion, for example when the recipient cells have been treated with R6G and concomitantly have a finite probability of escaping the mitochondrial inactivation and forming non-cybrid colonies. In the case of mES cybrids, it is also useful to be able to monitor for the presence of contaminating feeder-cell nDNA. This can be done using simple PCR assays involving two independent microsatellite markers that together permit identification of nDNA of strain 129 mES cells, either SNL76/7 or STO feeder cells, and the LA9 fibroblast cell line.

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