Genetic Manipulation 1 Flashcards
Pluripotency
The ability of a cell to contribute to any tissue in the body
pluripotent teratoma examples
sacrococcygeal teratomas in males 1 in 35 000
ovarian teratoma
what are teratomas?
tumours formed by germ cells that form oocytes and sperm
ovarian teratomas
usually benign and contain differentiated tissues
testicular teratocarcinoma
EC cells which are pluripotent
Embryonic stem cells
Pluripotent stem cells derived from inner cell mass of blastocyst of pre-implantation embryos
how many cells in the pre-implanted blastocyst?
50-150
Who discovered ES cells?
martin evans and matthew kaufman
What can you find out about ES cells in culture?
check expression of ICM/stem cell genes oct 4 and nanog for pluripotency
cell surface markers eg SSEA1
check sex and karyotype - SRY and HPRT
What is LIF used for?
leukaemia inhibiting factor prevents ES cell differentiation
What happens when ES cells are introduced into an adult mouse?
form teratomas
what happens when ES cells are re-introduced into a blastocyst?
contribute to all parts of embryo and form a chimeric mouse
oct 4
transcription factor expressed in ICM cells
what happens if there are no oct 4 genes?
develop into blastocyst but then die
what happens if there are no nanog genes?
lose pluripotency and develops extra embryonic tissue - parietal endoderm
What happens if LIF is withdrawn from ES cells grown on dishes or in suspension?
form ball like embryoid bodies
embryoid bodies
contain differentiating cells of 3 embryonic lineages and are trying to form an embryo
what may embryoid bodies also contain?
cardiomyocytes that can beat
How can we get fully GM mice from ES cells?
genetically manipulate cells - mutation/new genes
reintroduce into mice - inject into ICM
chimeric mice - breed these together
How is DNA introduced into cells?
bathe cells in media containing the DNA and pass shock through them - electroporation
Electroporation
introduced DNA into cells by making small holes in the cell membrane
What is done to the female mouse before implanting the ES cells?
mated with vasectomised mouse to allow uterus to swell and vascularise?
How to tell the difference between host blastocyst and donor ES cells
different strains of mice and different coat colours
