1. Introduction Embr...

نتيجة التلخيص (50%)

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).

النص الأصلي

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.

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