Commonly used ES Cell Lines

There are few commercial sources of ES cells. Those that exist at the time of publication are available through Lexicon (The Woodlands, TX; www.lexicon-genetics.com), Primogenix (St. Louis, MO), Specialty Media (Phillipsburg, NJ; www.specialtymedia.com) and ATCC (Manassas, VA; www.atcc.org). Of stem cell lines available, each may have its own characteristics (see Table 11.4). ES cells are best used at early passage because the cells may acquire spontaneously arising genetic mutations when kept in culture. As a guide, try to obtain cells for experimental use at passage ten or below. If you wish to establish a bank of cells for use in multiple experiments, then try to obtain a vial at about passage five, so that you can expand the cells and freeze them at passage seven to ten for use in

Mice E14 Overy
Fig. 11.6. Photograph of rat ovary and corpora lutea. Note approximately 50% of ovary obscured by fat pad

individual experiments. The ultimate solution would be to develop one's own ES cell line, by culturing from blastocysts. This is ideal because you will have first-hand access to the lowest passage cells possible. Also, it has been reported that ES cells perform best if kept in consistent culture conditions. Any change in culture conditions, such as might occur by transfer to another laboratory, has the potential to reduce the pleuripotency of ES cells. For example, differences in water quality used to make media, as well as differences in the source and condition of supporting fibrob-last cell lines, may reduce the capacity of the cells to transmit through the germline. Even a switch between two otherwise superb feeder cell lines has been purported to reduce ES cell potential.

The culture and use of ES cells is a specialized field and it is not our goal to include that information here. Further detailed reading can be found in several recommended texts (Wurst and Joyner 1995; Torres and Kuhn 1997; Abbondanzo et al. 1993; Doetschman 1994; Robertson 1987; Nagy et al. 2003). Most ES cells available today originate from one of several substrains of the 129 inbred mouse. Because of a pre-disposition towards

Table 11.4. Commonly used embryonic stem cell lines

Embryonic stem cell line

Originator

Parental mouse strain

Genes for coat color at A, B, C and P loci

Characteristics

EKCCE

M. Evans

129SvEv

AABBCC

E14

E. Robertson

129/Ola

AABBcchcchpp

HM1

D. Melton

129/Ola

AABBcchcchpp

Feeder free

AB-1 and AB-2

A. Bradley

129SvEvBrd

AABBCC

Hprt deficient

J1

R. Jaenisch

129SvJae

AABBCC

D3

T. Doetchman

129SvPas

AABBCC

R1

R. Jaenisch

129SvJ x

AABBCcchPp

Aggregation

129Sv-+p

chimera competent

See Table 3.1B,

R. Jaenisch

B6129

AaBBCC

Tetraploid embryo

Chap. 3,

complementation

this manual

competent

Bruce4

C. Stewart

C57BL/6

aaBBCC

naturally occurring teratocarcinomas, the 129 mouse strain facilitated the establishment of embryonal carcinoma (EC) cell lines - a cell type morphologically similar to ES cells and capable of differentiating into various cell types under specific culture conditions; albeit in a more limited capacity than ES cells. Despite their more restricted use, research on 129-derived EC cell lines paved the way for the development of ES cell technology, now commonly used in gene targeting experiments in mice.

Currently, many of the most widely used pleuripotent ES cell lines available are derived from strains of the mouse line 129. However, the C57BL/6 mouse strain is the best characterized scientifically, and therefore many researchers consider it necessary to backcross their mouse model onto a C57BL/6 background. This can take up to 2 years, since it is necessary to go though ten generations of backcrossing before reaching a genetic background closely approximating to C57BL/6. More recently, C57BL/6 ES cells have become available and although they do not perform with the same efficiency as 129 derived lines (Seong et al. 2004), there is an obvious advantage in putting a mutation directly into this genetic background. In addition, various hybrid ES cells derived from matings between either two different 129 substrains, or from two completely different inbred strains now exist. These hybrid cell lines are proving to be capable of giving rise to animals that are wholly of ES cell origin, in the founder generation (see Chap. 3, Generation of ES Cell-Derived Embryos and Mice by Tetraploid-Embryo Complementation by K. Eggan and R. Jaenisch).

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