Genetic Manipulation pt1 Flashcards
pluripotency
ability of a cell to contribute to any tissue in the body
pluripotent teratocarcinomas
“germ cell cancers”
tumours formed by germ cells (oocyte/sperm)
embryonal carcinoma cells
EC cells
cells of testicular teratocarcinomas, can be kept in culture
e.g. F9 cells
teratocarcinoma cells are pluripotent, proven by?
- if take cells & transplant under skin of immunodeficient host mouse, they form tumour of various tissue types
- if introduce F9 cells into mouse blastocyst in vitro and implant into pseudopregnant mouse they will give birth to chimeric offspring which have contributions from original blastocyst and EC cells
ES cells can be derived from…
dissociating a blastocyst and culturing the ICM
how do we know ES cells are pluripotent
can be made to differentiate into many cell types in culture
will form teratomas if introduced into adult mice
will contribute to all parts of embryo if reintroduced into blastocyst
pluripotency genes
Oct4:
transcription factor expressed in ICM cells
Oct4 null embryos develop to blastocyst but then die as ICM not pluripotent
Nanog:
expressed in ICM cells
Nanog-null ICM cells lose pluripotency and develop extra-embryonic tissues (parietal ectoderm)
how to maintain pluripotency of ES cells in culture
need leukaemia inhibitory factor (LIF):
signals through signalling pathways (JAK-STAT, Map kinase, PI3-kinase) to maintain Oct4 expression and prevent differentiation to mesoderm/endoderm
Bone morphogenic protein (BMP):
prevents differentiation into neuroectoderm
what happens if withdraw LIF from ES cells on culture
embryoid bodies form - contain differentiating cells
genetic manipulation of ES cells
ES cells can be genetically manipulated in vitro and then introduced into mouse blastocyst which can be implanted into pseudopregnant female mouse
have chimeric offspring which can then breed to produce fully genetically modified offspring
electroporation
cells bathed in solution of new gene
electrocourette gives electric shock, blowing tiny lesions in cell membrane. they heal in milliseconds but gives time for new DNA to enter and get to nucleus
reasons why genetic manipulation is done
understand basis of human, health & disease
identify the role of specific genes in disease
understand what drives been regulation
create designer animals for research, medical or other uses e.g. disease models
examples of useful spontaneous mutations
Pax6 mutation: pax6 is a transcription factor, mice has small eyes, important for eye and brain development
Vangl2 mutation: important for neural tube closure, mice without fail to develop proper tails
Limk1 mutation: Limk1 is an axon guidance related enzyme. sciatic nerve doesn’t growth properly in this mutation
random mutagenesis: how
exposing animals to a chemical mutagen
ENU (ethylnitrosourea):
creates point mutations by ethylating DNA pairs during DNA replication in sperm cells
EMS (ethyl methanesulphonate):
turns G/C pairs to A/T during DNA replication
mutagenesis screens
mate mutated males with WT females
offspring heterozygous for dominant gene will show a phenotype
for recessive, mate offspring to create homozygous babies
homologous recombination
usually occurs during meiosis
maternal and paternal copy of each chromosome line up and identical sequences of DNA find eachother and cross over
can happen anytime in cells when identical DNA sequences find eacoterh and line up
can be used to introduce new DNA into cells
gene knockout by homologous recombination
flank new gene with DNA that surrounds current gene. The target vector should also have a identifier gene (neomycin resistance, GFP) and thymidine kinase gene outside the flanking sequences.
electroporate target vector into ES cells
add neomycin, cells that haven’t taken up the DNA will die
retainment of TK gene means the new DNA was randomly integrated but if TK is not taken up and remains on target vector then HR occurred –> five ganciclovir to kill cells with TK
do PCR
inject new ES cells into ICM of host blastocyst , implant that into female mouse –> chimeric offspring –> breed offspring to create homozygous knockouts
transgenic animals
contain an exogenous gene introduced artificially
generally all cells contain the transgender and genetic change is heritable
GM: can make animals that..
contain mutant forms of gene of interest
overexpress gene of interest, or express it in different tissues
express important products e.g. insulin in milk
express genetic markers e.g. GFP to study control of gene expression
building a transgene - what a gene needs to work…
promoter sequence driving expression in appropriate tissue (transcription factors bind)
open reading frame encoding gene want expressed and bit encoding amino acids that line up to form protein
sequences that ensure correct mRNA processing e.g. polyadenylation signal so mRNA gets polyadenylated tail
different methods to introduce trans genes into animal
direct injection DNA
chemical transfection
electroporation
viral infection
chemical transfection of DNA
incubate cells in culture medium containing DNA and a chemical that wraps up the DNA
will either endocytose or diffuse through cell membrane
how to increase chance that all cells of transgenic animal will carry the transgene
introduce transgene into the one cell embryo
(if inject into one cell of an 8 cell embryo, get a mosaic)
direct injection of DNA
inject DNA into male pronucleus before it fuses with the female one
DNA repair mechanisms will recognise free DNA and hopefully integrate it into host DNA
direct injection of DNA: integration of transgene into injection
integration random
DNA may be chewed up by repair enzymes instead
may put DNA into different gene
direct injection of DNA: what if expression is not as expected
weak promoter/insufficient regulatory sequences
copy number - don’t know how many copies going in
site of integration - DNA may be inserted near another gene and be controlled by that genes promoter
epigenetic modification - cells think new DNA is viral & shut it down
3 genes needed for virus to infect and make more virus s
gag - encodes proteins of nucleoprotein core
pol - encodes reverse transcriptase and integrase
Env - encodes surface protein components of virion
packaging signal allows it to be wrapped up in new viral particle
how to use a retrovirus to get transgene into animal cells
genetically engineer transgene to look like retrovirus - retroviral flanking sequences and packaging signal
introduce this to helper cells, which contain genes encoding gag, pol and env, in vitro
now have new viral particles made with transgene RNA –> can be used to infect cells
cons of viral delivery
may only work in dividing cells
cells spot virus and shut them down , longterm expression is a problem
side effects (recombination leads to infective new viruses) safety considerations
viruses are excellent way to introduce genes to cells in culture or to particular tissues of an animal in vivo
not a good way of getting DNA integrated into whole animal (often silneced)
