Lakhasly

1.In addition, healthy cells growing in log phase are critical for optimal transformation efficiency in gene targeting experiments.The criteria used in our laboratory to qualify an ES cell clone for making chimeras is that at least 50% of the chromosome spreads analyzed must be 40 XY. In our experience, our DBA/1LacJ ES cells (12) meet or exceed that criterion Murine Embryonic Stem Cells 1 at least 86% of the time, whereas our 129 strain of ES cells meet or exceed the criteria 45% of the time.Smithies and colleagues later demonstrated that ES cells, modified by gene targeting when reintroduced into blastocysts, could transmit the genetic modifications through the germline (7).ES cells have been reported for other mammalian species (i.e., hamster, rat, mink, pig, and cow), however, only murine ES cells have successfully transmitted the ES cell genome through the germline.ES cells are isolated from the inner cell mass (ICM) of the blastocyst stage embryo and, if maintained in optimal conditions, will continue to grow indefinitely in an undifferentiated diploid state.ES cells that are not cared for properly will spontaneously differentiate, even in the presence of feeder layers and leukemia inhibitory factor (LIF).Today, genetic modification of the murine genome by ES cell technology is a seminal approach to understanding the function of mammalian genes in vivo.(1-4).

1. Introduction
   Embryonic stem (ES) cells were first isolated in the 1980s by several independent
   groups. (1–4). These investigators recognized the pluripotential nature of ES cells
   to differentiate into cell types of all three primary germ lineages. Gossler et al. (5)
   described the ability and advantages of using ES cells to produce transgenic animals
   (5). The next year, Thomas and Capecchi reported the ability to alter the genome of the
   ES cells by homologous recombination (6). Smithies and colleagues later demonstrated
   that ES cells, modified by gene targeting when reintroduced into blastocysts, could
   transmit the genetic modifications through the germline (7). Today, genetic modification
   of the murine genome by ES cell technology is a seminal approach to understanding the
   function of mammalian genes in vivo. ES cells have been reported for other mammalian
   species (i.e., hamster, rat, mink, pig, and cow), however, only murine ES cells have
   successfully transmitted the ES cell genome through the germline. Recently, interest
   in stem cell technology has intensified with the reporting of the isolation of primate
   and human ES cells (8–11).
   ES cells are isolated from the inner cell mass (ICM) of the blastocyst stage embryo
   and, if maintained in optimal conditions, will continue to grow indefinitely in an
   undifferentiated diploid state. ES cells are sensitive to pH changes, overcrowding, and
   temperature changes, making it imperative to care for these cells daily. ES cells that are
   not cared for properly will spontaneously differentiate, even in the presence of feeder
   layers and leukemia inhibitory factor (LIF). In addition, healthy cells growing in log
   phase are critical for optimal transformation efficiency in gene targeting experiments.
   Targeted murine ES cells have little value if they lose the ability to transmit the
   introduced mutations through the germline of the resulting chimeras. Therefore, it is
   critical that murine ES cells have a normal 40 XY karyotype. It is standard practice in
   our laboratory to have complete karyotypic analysis of all targeted ES cells prior to the
   production of chimeras. The criteria used in our laboratory to qualify an ES cell clone
   for making chimeras is that at least 50% of the chromosome spreads analyzed must be
   40 XY. In our experience, our DBA/1LacJ ES cells (12) meet or exceed that criterion
   Murine Embryonic Stem Cells 1
   at least 86% of the time, whereas our 129 strain of ES cells meet or exceed the criteria
   45% of the time.
   The many opportunities that exist in stem cell biology today, combined with the
   need to further explore and develop new technologies, makes it necessary to clearly
   define the process of developing stem cell lines. Therefore, this chapter will present
   the methods used in our laboratory to develop murine ES cell lines and maintain them
   in an undifferentiated state.