Mammalian development, genetic manipulation and
stem cells
1. Recap of mammalian development
Pre-implantation human/mouse development (mouse and human development is
very similar)
Gastrulation, Neurulation, Organogenesis
2. Tools to study mammalian development
- Studying mouse development:
Genetic screens not suitable: larger genome, longer generation time, adults
bigger, very expensive
Experimental embryology not suitable: embryos smaller, development inside
mother
Can use powerful genetic modifications:
a) Transgenics – add gene of interest
- Example of reverse genetics
- Method of overexpressing a gene product in Xenopus: Inject mRNA into the
embryo, lots of protein is then made. Problem: the mRNA is gradually degraded.
Not heritable.
- Method of overexpressing a gene product in mouse (transgenics):
Fertilized zygotes are removed from a female mouse and incubated in a dish.
To inject plasmid DNA into the zygotes, first they must be held still using a
holding pipette. A very fine injection pipette containing the DNA is then inserted
into one of the pronuclei (this is usually always the male pronuclei which is the
larger of the two).
When the solution containing the DNA is expelled the pronuclei swells slightly.
The zygotes are then transferred into the oviduct of a pseudopregant recipient (a
female mouse that is in the correct stage of the estrous cycle and has mated
with a non-fertile male the previous night. The DNA can integrate into the
genome in a random location (or in most cases, not at all).
The mice that are born need to be screened to see which contain the transgene.
Easy identification method for transgenic mice uses GFP expression in the
, expidermis, however most transgenics need to be screened by a PCR reaction to
detect the endogenous DNA sequence.
b) Targeted knockouts – remove gene of interest
Example of reverse genetics
Obtain ES cells (embryonic stem cells) to genetically alter
Make a targeting vector
Genetically modify ES cells – add complementary DNA sequence which removes
section of DNA and inserts into genome in its place
Inject modified ES cells into blastocyst stage embryos
Implant recombinant embryos into a surrogate mother
Breed the chimeric pups
- Method of removing a gene of interest in mouse (targeted knockouts):
In order to create a mouse knockout you first of all need undifferentiated mouse
ES cells growing in culture. ES cells need to be taken from the inner cell mass. A
stem cell can produce more stem cells or differentiated cells – ES cells need very
carefully regulated growing conditions to stop them from differentiating.
- How to genetically modify ES cells:
1. Firstly, construct a plasmid called a targeting vector. This is made up of
pieces of DNA ‘stitched’ together using molecular biology techniques (such as
the use of restriction enzymes). These DNA sequences are contained within
plasmids and can therefore be replicated in bacteria that contain the plasmid – 4
key parts:
a) Homologous arms flanking gene
b) Positive selectable marker e.g. neomycin resistance gene
c) Negative selectable marker e.g. thymidine kinase
d) Reporter gene e.g. B-galactosidase
stem cells
1. Recap of mammalian development
Pre-implantation human/mouse development (mouse and human development is
very similar)
Gastrulation, Neurulation, Organogenesis
2. Tools to study mammalian development
- Studying mouse development:
Genetic screens not suitable: larger genome, longer generation time, adults
bigger, very expensive
Experimental embryology not suitable: embryos smaller, development inside
mother
Can use powerful genetic modifications:
a) Transgenics – add gene of interest
- Example of reverse genetics
- Method of overexpressing a gene product in Xenopus: Inject mRNA into the
embryo, lots of protein is then made. Problem: the mRNA is gradually degraded.
Not heritable.
- Method of overexpressing a gene product in mouse (transgenics):
Fertilized zygotes are removed from a female mouse and incubated in a dish.
To inject plasmid DNA into the zygotes, first they must be held still using a
holding pipette. A very fine injection pipette containing the DNA is then inserted
into one of the pronuclei (this is usually always the male pronuclei which is the
larger of the two).
When the solution containing the DNA is expelled the pronuclei swells slightly.
The zygotes are then transferred into the oviduct of a pseudopregant recipient (a
female mouse that is in the correct stage of the estrous cycle and has mated
with a non-fertile male the previous night. The DNA can integrate into the
genome in a random location (or in most cases, not at all).
The mice that are born need to be screened to see which contain the transgene.
Easy identification method for transgenic mice uses GFP expression in the
, expidermis, however most transgenics need to be screened by a PCR reaction to
detect the endogenous DNA sequence.
b) Targeted knockouts – remove gene of interest
Example of reverse genetics
Obtain ES cells (embryonic stem cells) to genetically alter
Make a targeting vector
Genetically modify ES cells – add complementary DNA sequence which removes
section of DNA and inserts into genome in its place
Inject modified ES cells into blastocyst stage embryos
Implant recombinant embryos into a surrogate mother
Breed the chimeric pups
- Method of removing a gene of interest in mouse (targeted knockouts):
In order to create a mouse knockout you first of all need undifferentiated mouse
ES cells growing in culture. ES cells need to be taken from the inner cell mass. A
stem cell can produce more stem cells or differentiated cells – ES cells need very
carefully regulated growing conditions to stop them from differentiating.
- How to genetically modify ES cells:
1. Firstly, construct a plasmid called a targeting vector. This is made up of
pieces of DNA ‘stitched’ together using molecular biology techniques (such as
the use of restriction enzymes). These DNA sequences are contained within
plasmids and can therefore be replicated in bacteria that contain the plasmid – 4
key parts:
a) Homologous arms flanking gene
b) Positive selectable marker e.g. neomycin resistance gene
c) Negative selectable marker e.g. thymidine kinase
d) Reporter gene e.g. B-galactosidase
