Lecture 24 Flashcards Preview

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Flashcards in Lecture 24 Deck (17):
1

So far with yeast:

- 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

So far with aspergillus:

- Rapid, easy
- Cloning by complementation
- Non-homologous (nkuAdelta) and homologous integration

3

So far with adabidopsis and drosohpila:

- 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

Mouse experiments:

- Initially microinjection directly into the embryo was very inefficient
- The development of ES cell cultures was a major advance

5

Embryonic stem (ES) cells:

- Can be maintained in culture
- Form colonies
- Remain in undifferentiated state

6

Totipotent:

- Retain the ability to differentiate into all cell types

7

Steps for transforming ES:

- 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

Gene transfer in the Mouse:

- 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

Why is there so much non-homologous integration in the mouse compared to the yeast?

- 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

Is it even possible to consider a gene targeting approach in mice/mammalian systems?

- 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

Positive-negative selection for mammalian gene targeting (1/100):

- 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

p53 gene:

- 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

Testing this:

- 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

Loss of p53:

- 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

GAL4 transcription factor:

- 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

16

Is p53 an activator/capable as acting as a promoter?

- Fuse p53 coding region to Gal4 DNA binding domain
- Inserted into yeast
- A hybrid protein has an activator function in yeast
- Attaching p53 to GAL4 promotes the activation of GAL4

17

p53:

- The guardian of the genome
- Required to control expression of DNA damage repair systems
- Without p53 these mechanisms don't operate and cancers and malignancy develop