Lecture 24 Flashcards
1
Q
So far with yeast:
A
- Rapid, easy and a range of vectors
- Large numbers of transformants means cloning by complementation is possible
- Homologous integration or autonomous replication
- Gene inactivation or modification availabl
2
Q
So far with aspergillus:
A
- Rapid, easy
- Cloning by complementation
- Non-homologous (nkuAdelta) and homologous integration
3
Q
So far with adabidopsis and drosohpila:
A
- Integration using agrobacterium Ti plasmid (plants) or P-element transposase (drosophila), not by homology
- Frequency too low for cloning by complementation
0- Can introduce any sequence, eg) reporter genes, enhancer trap - Gene inactivation difficult, knock down by RNAi
4
Q
Mouse experiments:
A
- Initially microinjection directly into the embryo was very inefficient
- The development of ES cell cultures was a major advance
5
Q
Embryonic stem (ES) cells:
A
- Can be maintained in culture
- Form colonies
- Remain in undifferentiated state
6
Q
Totipotent:
A
- Retain the ability to differentiate into all cell types
7
Q
Steps for transforming ES:
A
- Add DNA with selectable marker
- Select for the cell that have taken up the DNA
- Micro inject ES cells into developing embryo
- Implant the embryo (carrying genes of interest) into a pseudo pregnant female
- Chimeric mouse is born (visualised by a white pseudo pregnant mouse and a brown ES cell)
- Cross chimeric with white, and uniformly brown offspring will show you a heterozygote
- Breed the heterozygotes together to generate a homozygote,
8
Q
Gene transfer in the Mouse:
A
- Labour intensive and low frequency
- Not large numbers produced
- Fate of DNA, must be integrated to be maintain
- Most of the integrations will be non-homologous which can be turned into homolgous
9
Q
Why is there so much non-homologous integration in the mouse compared to the yeast?
A
- Is it due to the amount of DNA in the mice? NO!
- This was tested using yeast and Leu2 gene
- Also tested with CHO cells and the DHFR gene
10
Q
Is it even possible to consider a gene targeting approach in mice/mammalian systems?
A
- Targeted disruption of HPRT (purine salvage enzyme), with NEO resistance selectable marker
- Select for neoR and 6-TGR (toxic to normal cells, but if hprt- they can’t generate the toxic compound)
- Selection needs to be independent of the target gene
- hprt gene is x linked
11
Q
Positive-negative selection for mammalian gene targeting (1/100):
A
- Positive for transformants (eg. NeoR)
- Negative for non-homologous integration events (eg. HSV-TK, GANCs)
- HSV-TK metabolises GANC into a toxic compound
- This works for any gene
- If integration is non-homologous the cells can be removed, so the cells you are looking at are only homologous inactivation of your gene of interest
12
Q
p53 gene:
A
- Role in cell cycle
- Alteration to p53 gene associated with many cancers
- Is this due to loss of function OR over expression/altered function? OR the effect of a null mutation?
13
Q
Testing this:
A
- Create a p53 construct containing HSV-TK and neoR, insertion of neoR will destroy the function of p53
- Selected NEOr GANCr ES clones
- Confirm homologous integration by southern blot (DNA)
- Heterozygous p53+/p53nulls were isolated after breeding out chimerics
- Cross heterozygotes with heterozygotes to get mendelian ratios of offspring
14
Q
Loss of p53:
A
- Malignancy
- An increase in cancers in p53 loss mice
- so p53 is suggested to have a protective role
- Does p53 activate expression of protective genes, or repress expression of genes that cause cancer
- Yeast can determine whether p53 can function as an activator of gene expression
15
Q
GAL4 transcription factor:
A
- in WT regulates galactose breakdown in yeast to utilise as a carbon source
- GAL4 has a DNA binding domain and an activation domain which can be separated
