Lecture 24

Question 1

So far with yeast:

Answer 1

• 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

Question 2

So far with aspergillus:

Answer 2

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

Question 3

So far with adabidopsis and drosohpila:

Answer 3

• 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

Question 4

Mouse experiments:

Answer 4

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

Question 5

Embryonic stem (ES) cells:

Answer 5

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

Question 6

Totipotent:

Answer 6

• Retain the ability to differentiate into all cell types

Question 7

Steps for transforming ES:

Answer 7

• 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,

Question 8

Gene transfer in the Mouse:

Answer 8

• 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

Question 9

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

Answer 9

• 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

Question 10

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

Answer 10

• 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

Question 11

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

Answer 11

• 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

Question 12

p53 gene:

Answer 12

• 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?

Question 13

Testing this:

Answer 13

• 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

Question 14

Loss of p53:

Answer 14

• 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

Question 15

GAL4 transcription factor:

Answer 15

• 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

Question 16

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

Answer 16

• 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

Question 17

p53:

Answer 17

• 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