Engineered embryonic stem cell method

1. THE ENGINEERED
EMBRYONIC STEM CELL
METHOD (STEPS)
P:3 U:2
Vedanti S. Gharat
T.Y. B.Sc. Biotechnology
Roll No. : 20

2. INTRODUCTION
 Transgenic technology has been developed and perfected in the laboratory
mouse. Since the early 1980s, hundreds of different genes have been
introduced into various mouse strains.
 These studies have contributed to an understanding of gene regulation,
tumor development, immunological specificity, molecular genetics of
development, and many other biological processes of fundamental interest.
 Transgenic mice have also played a role in examining the feasibility of the
industrial production of human therapeutic drugs by domesticated animals
and in the creation of transgenic strains that act as biomedical models for
various human genetic diseases.
 For transgenesis, DNA can be introduced into mice by
(1) retroviral vectors, that infect the cells of an early-stage embryo prior to
implantation into a receptive female
(2) microinjection, into the enlarged sperm nucleus (male pronucleus) of a
fertilized egg
(3) introduction of genetically engineered embryonic stem cells into an early-
stage developing embryo before implantation into a receptive female.

3. ENGINEERED EMBRYONIC
STEM CELL METHOD
Embryonic Stem Cells (ESCs)
are stem cells derived from
the undifferentiated inner
mass cells of a human
embryo.
Embryonic Stem Cells are
Pluripotent – they are able
to grow (i.e. differentiate)
into all derivatives of the
three primary germ layers :
Ectoderm, Endoderm and
Mesoderm.

4. ENGINEERED EMBRYONIC
STEM CELL METHOD
Cells from the early,
blastocyst stage of a
developing mouse embryo
can proliferate in cell
culture and still retain the
capability to differentiate
into all other cell types—
including germ line cells—
after they are reintroduced
into another blastocyst
embryo. Such cells are
called Pluripotent
embryonic stem cells.

5. ENGINEERED EMBRYONIC
STEM CELL METHOD
 When in culture, embryonic stem cells can be readily engineered
genetically without altering their pluripotency.
 With this system, a functional transgene can be integrated at
a specific site within a dispensable region of the genome of
embryonic stem cells.
 The genetically engineered cells can be selected, grown, and used to
generate transgenic animals.
 In this way, the randomness of integration that is inherent in the
DNA microinjection and retroviral vector systems is avoided.

6. Establishing transgenic mice with
genetically engineered embryonic
stem(ES) cells. An embryonic
stem cell culture is initiated from
the inner cell mass of a
mouse blastocyst. The embryonic
stem cells are transfected with a
transgene. After growth, t he
transfected cells are identified by
either the positive-negative
selection procedure or PCR
analysis. Populations of
transfected cells can be cultured
and inserted into blastocysts,
which are then implanted into
foster mothers. Transgenic lines
can be established by crosses from
founder mice that carry the
transgene in their germ lines.

7. ENGINEERED EMBRYONIC
STEM CELL METHOD
 Embryonic stem cells carrying an integrated transgene can be
cultured and inserted into blastocyst stage embryos, and these
embryos can then be implanted into pseudo pregnant foster mothers.
 Transgenic lines are established by mating the progeny that carry
the transgene in their germ lines.
 Then, if required, littermates that carry a transgene in their germ
lines are crossed to produce mice that are homozygous for the
transgene.

8.  After transfection of embryonic stem cells in culture with a DNA vector
that is designed to integrate within a specific chromosomal location, some
cells will have DNA integrated at non target (spurious) sites, whereas in
other cells, integration will occur at the target (correct) site.
 The target site should be located in a section of genomic DNA that encodes
no essential products, so that after integration of the input DNA, there is no
interference with any developmental or cellular functions.
 Moreover, it is essential that the transgene be integrated into a part of the
genome that does not prevent it from being transcribed, for example, in
euchromatin rather than hetero-chromatin.
 In most of the embryonic stem cells, the input DNA will not be integrated
at all. To enrich for the cells with DNA integrated at the target site, a
procedure called positive-negative selection is implemented.
 This strategy uses positive selection for cells that have vector DNA
integrated anywhere in their genomes and negative selection against the
vector DNA sequence that is integrated at spurious sites.

9. ENGINEERED EMBRYONIC
STEM CELL METHOD
 Not only can a transgene be inserted into a specific chromosome
site by homologous recombination in embryonic stem cells to
provide a new function, but a specific mouse gene can also be
targeted for disruption by the incorporation of a DNA sequence,
usually a selectable marker gene, into its coding region.
 One of the aims of targeted gene disruption (gene knockout) is to
determine the developmental and physiological consequences of
inactivating a particular gene.
 In addition, a transgenic line with a specific disabled gene can
be used as a model system to study the molecular pathology of a
human disease.

10. BIBLIOGRAPHY:-
Molecular Biotechnology: Principles and Applications of
Recombinant DNA 4th Edition by Glick, Pasternak and
Patten.