Lecture 20 - Reproductive Manipulation And The Routes For Transgenesis

Question 1

Stages of fertilisation

Answer 1

Egg divides creating a morela and then a blastocyst before implanting in the mother.

Question 2

Fertilisation and timing of preimplantation events?

Answer 2

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

Question 3

What is trans genesis

Answer 3

Is a controlled transfer of genetic information into the genome and the DNA can be taken from different species or synthesised

Question 4

What are the 2 ways to generate transgenic animals?

Answer 4

Injection of DNA into pro nucleus of egg

Transfection of embryonic stem cells with DNA, select for genetic change and reintroduce cells into embryo

Question 5

What is pronuclear trans genesis?

Answer 5

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

Question 6

Where do we get transgenes from?

Answer 6

Purified DNA from plasmid, BAC or yAc vectors

Question 7

Why do we use transgenes?

Answer 7

To express genes or to analyse their regulatory elements

Question 8

What is the set up for pronuclear trans genesis?

Answer 8

Inverted microscope
Micromanipulator mounted Nettie pressure holding pipettes
Micromanipulator mounted injection pipettes

Question 9

How do you make a pro nucleus?

Answer 9

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

Question 10

The pathway of getting trans genesis eggs to mice?

Answer 10

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

Question 11

Why do you want superovulation?

Answer 11

Not all eggs will take the DNA and many die after fertilisation

Question 12

What is position effect?

Answer 12

Injected DNA integrates at random chromosomal locations. Site of integration often influences expression of the transgene

Question 13

Can integration of te transgene be mutagenic?

Answer 13

Yes

Question 14

Does injected DNA recombines to form concatemers before integration? And what are these usually?

Answer 14

Yes and they are usually multicopy arrays

Question 15

Why would transgene insertions not always be intact?

Answer 15

Injected DNA may be partially degraded before integration

Question 16

What makes the transgenic line unique?

Answer 16

Integration site
Copy number
Integrity of transgene

Question 17

What can happen to a gene during pronuclear trans genesis?

Answer 17

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

Question 18

Generating target mutations in mice - is early mammalian embryogenesis regulation?

Answer 18

Yes

Question 19

How do you make a aggregation chimera?

Answer 19

You take two implanted embryos and combine them to produce a single animal

Question 20

What are the 3 key cells of the blastocyst and what do they do?

Answer 20

Epiblasts - all parts of animal body
Hypoblasts - yolk sac
Trophoblast - placenta

Question 21

What are epiblast cells - are they pluripotent and what do they make

Answer 21

Yes

Ectoderm, mesoderm, endoderm

Question 22

Where are ES cells derived from?

Answer 22

Epiblasts

Question 23

What happens if you inject ES cells back into the blastocyst?

Answer 23

They can make any animal cell

Question 24

What can Epiblasts and ES cells give rise to?

Answer 24

Tumours when injected elsewhere (called teratocrcinomas)

Question 25

How do you make a mutant mouse through ES cells?

Answer 25

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

Question 26

How do you make a chimera using stem cells?

Answer 26

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.

Question 27

How do you generate cells with targeted mutations?

Answer 27

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

Question 28

How do you generate cells with targeted mutations - what are the 2 ways this can be done?

Answer 28

Gene targeting and random integration

Question 29

How do you generate cells with targeted mutations - how do you do gene targeting?

Answer 29

Homologous recombination which means you can select for cells which have one gene and not the other

Question 30

How do you generate cells with targeted mutations - random integration

Answer 30

Non homologous recombination so all target set (vector you had made) is integrated meaning that the cells will survive

Question 31

What happens if the length of homologous arms to chromosomal locus increases?

Answer 31

Targeting frequency increases

Question 32

What construct shows greater targeting efficiency than non-isogenic constructs?

Answer 32

Isogenic constructs

Question 33

What would using non-isogenic DNA cause?

Answer 33

Mismatch’s, Msh2 protein recognises these and destabilises heteroduplex DNA formation

Question 34

What is isogenic?

Answer 34

Same mouse strain

Question 35

How do you set up a gene targeting experiment?

Answer 35

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

Question 36

How do you do a southern blot in gene targeting - what does it require?

Answer 36

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

Question 37

How do you do a PCR for screening targeted clones?

Answer 37

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

Question 38

How do you analyse gene expression pattern using reporter constructs?

Answer 38

Can be achieved by insertion of the reporter into the locus by targeting

Question 39

What is a knock in vector?

Answer 39

CDNA can be inserted this way to express a gene under the control of an endogenous genes

Question 40

What is an example of gene expression using reporter constructs?

Answer 40

GFP knock in Mouse which goes flourescent

Question 41

What are multicistronic constructs?

Answer 41

Constructs which can do multiple genes at once

Question 42

What are the two ways you can make a multicistronic construct?

Answer 42

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

Question 43

Where can reporters be localised to and what are these called?

Answer 43

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

Question 44

What are the two possibilities of homozygous gene targeting?

Answer 44

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)

Question 45

Most integrations are random but it is possible to do what? In es cell targeted genetic manipulation?

Answer 45

to selectively enrich for and identify even rare clones in which the construct has integrated into the target gene

Question 46

Can you use homozygous ES cell lines?

Answer 46

Yes

Question 47

What ones targeted nock out mutation in Runx1 gene result in?

Answer 47

Haemorrhage and anaemia

Question 48

What does lacZ reporter incorporated in Hox11 targeted mice precisely pinpoint?

Answer 48

asplenic phenotype in -/- embryos

Question 49

Do genes sometimes need to be disrupted to reveal full mutant phenotype - example?

Answer 49

Yes - Hox10 paralogue

Question 50

Why would some mutations not show?

Answer 50

Genetic redundancy = other genes are over compensating for it - this can be assessed for gene overexpression.