Lecture 20 - Reproductive Manipulation And The Routes For Transgenesis Flashcards
Stages of fertilisation
Egg divides creating a morela and then a blastocyst before implanting in the mother.
Fertilisation and timing of preimplantation events?
Pre-implantation development proceeds in culture normally and cultured embryos can be transferred into the oviduct or uterus of a pseudo pregnant female and will develop into normal mice
What is trans genesis
Is a controlled transfer of genetic information into the genome and the DNA can be taken from different species or synthesised
What are the 2 ways to generate transgenic animals?
Injection of DNA into pro nucleus of egg
Transfection of embryonic stem cells with DNA, select for genetic change and reintroduce cells into embryo
What is pronuclear trans genesis?
Controlled transfer of genetic information into the genome from any source - synthetic or species and confers a large phenotypic change. There is no reliance on selective breeding
Where do we get transgenes from?
Purified DNA from plasmid, BAC or yAc vectors
Why do we use transgenes?
To express genes or to analyse their regulatory elements
What is the set up for pronuclear trans genesis?
Inverted microscope
Micromanipulator mounted Nettie pressure holding pipettes
Micromanipulator mounted injection pipettes
How do you make a pro nucleus?
Fertilised one-cell eggs for DNA micro injection are obtained from superovulating female mice that have been mated to stud males. The male pro nucleus of a fertilised egg is injected wit politer amounts of purified DNA
The pathway of getting trans genesis eggs to mice?
Hormonal injects = superovulating female
Isolate fertilised eggs
Add the transgenes DNA
Implant injected eggs into pseudo pregnant foster female
Mice are born and their DNA is taken from their tails and PCR
Why do you want superovulation?
Not all eggs will take the DNA and many die after fertilisation
What is position effect?
Injected DNA integrates at random chromosomal locations. Site of integration often influences expression of the transgene
Can integration of te transgene be mutagenic?
Yes
Does injected DNA recombines to form concatemers before integration? And what are these usually?
Yes and they are usually multicopy arrays
Why would transgene insertions not always be intact?
Injected DNA may be partially degraded before integration
What makes the transgenic line unique?
Integration site
Copy number
Integrity of transgene
What can happen to a gene during pronuclear trans genesis?
1) Overexpression of gene - example - fat rats
2) defining regulatory elements by BAC scanning - example - lacZ inserted into BAC drive expression but different anatomical results
Generating target mutations in mice - is early mammalian embryogenesis regulation?
Yes
How do you make a aggregation chimera?
You take two implanted embryos and combine them to produce a single animal
What are the 3 key cells of the blastocyst and what do they do?
Epiblasts - all parts of animal body
Hypoblasts - yolk sac
Trophoblast - placenta
What are epiblast cells - are they pluripotent and what do they make
Yes
Ectoderm, mesoderm, endoderm
Where are ES cells derived from?
Epiblasts
What happens if you inject ES cells back into the blastocyst?
They can make any animal cell
What can Epiblasts and ES cells give rise to?
Tumours when injected elsewhere (called teratocrcinomas)
How do you make a mutant mouse through ES cells?
Get a pluripotent ES cell from Epiblasts of blastocyst embryos obtained from the uterus of a female mouse
Culture on plates containing non-dividing embryonic fibroblasts called feeder cells, foetal calf serum and LIF. (BMP4 + LIF can also be used)
The feeders keep the ES cells undifferentiated
How do you make a chimera using stem cells?
Isolate blastocyst from two different coloured mice and inject which one coloured mouse ES cells (agouti cells) and implant the chimeric blastocyst into pseudo-pregnant albino female mouse.
If you cross the chimeric mouse with a non-chimeric mouse they will have non-transgenic kids.
How do you generate cells with targeted mutations?
DNA vector is designed for homologous recombination with a designated gene. The genes which survive will have a Neo positive selection marker which confers resistance of to G418
How do you generate cells with targeted mutations - what are the 2 ways this can be done?
Gene targeting and random integration
How do you generate cells with targeted mutations - how do you do gene targeting?
Homologous recombination which means you can select for cells which have one gene and not the other
How do you generate cells with targeted mutations - random integration
Non homologous recombination so all target set (vector you had made) is integrated meaning that the cells will survive
What happens if the length of homologous arms to chromosomal locus increases?
Targeting frequency increases
What construct shows greater targeting efficiency than non-isogenic constructs?
Isogenic constructs
What would using non-isogenic DNA cause?
Mismatch’s, Msh2 protein recognises these and destabilises heteroduplex DNA formation
What is isogenic?
Same mouse strain
How do you set up a gene targeting experiment?
1) transfect cell with targeting construct
2) select cells and put onto a plate
3) drug resistant colonies will survive and grow
4) you make replica plates
5) one plate use to see if gene is there through a southern plot the other choose the correct genes for your experiment
How do you do a southern blot in gene targeting - what does it require?
requires the availability of suitable restriction sites in the target locus and probes for hybridisation that lie out-with the region encompassed by the homology arms
Restriction enzyme digestion will reveal a change in size of the endogenous fragments due to the insertion of new sequence revealed by Southern blot analysis and hybridisation with the probe
How do you do a PCR for screening targeted clones?
Can be designed with one primer complementary to the selection marker and the other primer complementary to the sequence of the target but out with the region encompassed by the homology arms
If targeting occurs these will amplify a defined predicted length of sequence, but if the reaction is properly optimised this amplification will not be detected in random integrations
How do you analyse gene expression pattern using reporter constructs?
Can be achieved by insertion of the reporter into the locus by targeting
What is a knock in vector?
CDNA can be inserted this way to express a gene under the control of an endogenous genes
What is an example of gene expression using reporter constructs?
GFP knock in Mouse which goes flourescent
What are multicistronic constructs?
Constructs which can do multiple genes at once
What are the two ways you can make a multicistronic construct?
IRES (internal ribosomal entry site) was initially isolated from viral DNA; later also found in cellular RNA. Allows CAP-independent translation. IRES normally attenuate expression of cDNA
2A peptides (also isolated from a virus) result in expression of multiplecistrons at equimolar levels
Where can reporters be localised to and what are these called?
Nuclear localisation signal - directs reporter to the nucleus
Myristoylation or faresylation signal sequence - anchor reporter to the cell membrane
Other sequences can be used to label mitochondria
What are the two possibilities of homozygous gene targeting?
1): two rounds of gene targeting using essentially the same vector but with different selection markers, e.g. first, neo vector (G418 selection) and then hyg vector (contains hygromycin selection marker
2): for neo resistant cassette: increased concentration of G418 gives both alleles targeted (this does not work for other selection markers)
Most integrations are random but it is possible to do what? In es cell targeted genetic manipulation?
to selectively enrich for and identify even rare clones in which the construct has integrated into the target gene
Can you use homozygous ES cell lines?
Yes
What ones targeted nock out mutation in Runx1 gene result in?
Haemorrhage and anaemia
What does lacZ reporter incorporated in Hox11 targeted mice precisely pinpoint?
asplenic phenotype in -/- embryos
Do genes sometimes need to be disrupted to reveal full mutant phenotype - example?
Yes - Hox10 paralogue
Why would some mutations not show?
Genetic redundancy = other genes are over compensating for it - this can be assessed for gene overexpression.
