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Flashcards in lecture 18 Deck (20)
1

What is transgenesis?

the process of transferring genes to animals

2

What is a transgenic animal?

an animal that carries a transgene

3

What is a transgene?

a foreign gene

4

What is a chimera?

an organism carrying cell populations from two or more different embryos of the same or different strains

- often use coat colour to see whether you have made your chimera

5

What does early mammalian development involve?

- fertilised egg
- division of blastomeres
- start to pack together tightly --> formation of morula - it pushes cells inside and it pushes cells on the outside, so you're left with some cells inside, starts to form these cells in the middle cells on the outside --> development of the blastocyst
- takes about 3/4 days in the mouse, 7 days in humans
- outside cells of the blastocyst are the trophoblast --> placenta
- inner cell mass = cells that form the embryo/human
- about 16 cells at the early stage
- before the blastocyst has implanted

6

How do we generate chimeras?

- you fertilise an egg in the oviduct and it travels down the oviduct while it's dividing
- once it gets to the uterus it is a blastocyst and it's waiting to implant into the uterine wall
- you can flush the oviducts before they've implanted and you can collect these blastocysts
- culture them and take the ICM cells and they expand in culture --> can go for 30 odd years
- it is when cells are in culture that they can be genetically modified, make alterations of our choice
- inject selected ES cells from black mouse into blastocyst from white mouse (into the blastoceal cavity)
- only have to add between 10 and 12 cells into that cavity in order to generate a chimera
- these cells will now contribute to the organism --> divide etc
- when injected into donor blastocyst, ES cells contribute to all tissues of the resulting offspring – models of human disease

7

What are the important steps in the gene-targeting techique?

a. generate the gene-targeting construct
- a piece of genome that you've extracted from the mouse, 2-3kb on either side of the region that you want to play with
- these side sequences are very important for driving homologous recombination
b. transfect construct into ES cells
- liposomes, electrophoration (most common, temporary holes in the membrane)
- can recombine and displace the region that is already there
c. select positively gene-targeted ES cells
- need to only take the cells that have taken up the construct
- doesn't work the same way as with mammal ES cells
- use other selectable markers, antibiotics in particular that have been generated for use in ES cells
- after a period of time only the colonies that have taken up the construct will grow
d. inject gene-targeted cells into blastocysts
e. transfer blastocysts to uterus of foster mothers
f. genotype offspring
g. cross heterozygotes to breed to homozygosity
- necessary to create a null mutation

8

What is homologous recombination?

- an exchange between similar genetic material

9

What is heterologous recombination?

- an exchange between different genetic material
- random integration

10

What are selectable markers for positive and negative selection?

- neomycin gene (NEO): G418 inhibits protein synthesis, Neo inactivates G418 (+)
- thymidine kinase gene (TK): ganciclovir nucleoside analog, TK converts ganciclovir into toxic product (-)
- flanking homologous sequences on either side, exon 1, neo, exon 1, TK downstream of flanking homologous sequence
- if you just put the neo cassette in you can't tell if it is homologous or non-homologous
- if you add TK and it's still present --> it will kill the cell that has taken it up
- only taken up in non-homologous recombination (not correct in all cases but a nifty trick)
- random integration - whole thing
- assumption is that in homologous recombination the whole construct does not go in --> the TK falls off

- non homologous will die in ganciclovir (even though resistant to G418)
- homologous --> Ganciclovir resistant --> cells live

11

What do you see in an analysis of DNA extracted from offspring following heterozygote crossing?

- gene locus containing gene mutation by insertion of neomycin gene is longer than for wildtype locus
- using PCR primers that bind to both ends of gene locus can amplify DNA
- primers are the same for mutated and Wildtype locus
- wt/wt would have one band, shorter
- wt/m two bands
- m/m one band (size of mutated locus)

12

What are knockouts?

- gene-targeting (precise location)
- loss of function
- null mutant
- no functional protein

13

What are knockins?

- gene-targeting
- modification
- may be functional protein
- may be other species gene (protein)

14

What are the applications of transgenics?

a. to study the function of a gene - create KO
b. generate models of human disease

does p53 have a role in cancer?
normal
- p53 levels very low
- prevents S-phase of cell cycle if DNA damaged

knockout p53 -/- mice
- mice normal, develop cancer by 3 months
- p53 mutations in human cancers
- model of human disease
- test new: drugs, vaccines, gene-therapy, etc

15

What if you create a KO and discover it has functions in many organs? How can you study the effect of the KO in one organ only?

tissue-specific gene-targeting

cre-lox system: tissue-specific gene-targeting
- removes DNA from between two specific sequences

Animal one: transgenic expresses Cre recombinase only in lung
- lung specific promoter/Cre recombinase gene
- therefore this enzyme is only expressed in the lung

Animal two: floxed gene created by gene targeting (flanked by LoxP site)

first two animals are totally normal
- cross the animals
- floxed gene meets Cre recombinase only in lung cells

16

What are applications of the cre-lox system?

normal:
Tcf 21 (Pod1, capsulin) - what is role?

KO - animals die within 5 mins of birth due to defects in lungs and other organs
- suggest Tcf21 has an important role in organ development
- can study the tissues to examine defects at a cellular level
- can study target genes by microarray or transcriptome analysis – this will allow you to understand what genes are turned on or off by Tcf21

since Tcf 21 -/- animals die within 5 mins, difficult to study specific role of Tcf21 in kidney development

generate two animals:
1. kidney specific promoter - Cre recombinase. promoter activare during late embryonic development (but could theoretically test at any stage of development with unique promoters)
2. floxed Tcf21

cross animals
- Tcf21 only KO in kidney
- can now study defect in kidney during late development

useful technique to study genes with multiple functions

these are conventional techniques

17

What is zinc finger nuclease technology ?

- advance
- method of the year 2011 – nature methods "genome editing with engineered nucleases"
- for its annual choice to highlight an important research method for biological researchers Nature Methods has selected "genome..." see Nat methods. 2011 Dec 28; 9(1):26
- in 2011 there were many high profile publications using zinc finger nucleases wiht a wide range of genomic edits in a variety of cell lines and species

zinc finger binding motif: zinc fingers are a type of protein structural motif that binds to DNA e.g. there are also leucine zippers, helix-loop-helix binding domains etc
- binds to DNA at three points
- important: specific amino acids on the proteins are specific for DNA sequences i.e. proteins do not bind randomly

- create two x two zinc finger proteins each with specificity for 12 nucleotides
- to each of the two finger motifs, attach a DNA nuclease - Fok 1
- when the DNA-binding and DNA-cleaving domains are fused together, a highly-specific pair of 'genomic scissors' are created
- a break can be made in a specific location anywhere in the genome
- can target where you want to make break in DNA by changing nucleotides on zinc finger proteins cDNA

- use expression vector to get zinc finger nuclease (ZFN) gene into cell of interest
- ZFN pair recognise and bind to target site
- ZFN makes double strand break
- the break stimulates DNA repair
a) 1-20% of cells are mis-repaired resulting in gene deletion
b) when repair template co-transfected with ZFN pair, 2-20% cells contain GOI at target site via homologous recombination

18

What are advantages of ZFN technology?

- For A) deletion mutation at, or B) integration into, any genomic location
- change is permanent and heritable
- works in many mammalian cells
- no selectable markers needed

19

what is ZFN gene-targeting technique?

- fertilised one-cell embryo
- microinjection of ZFN into nucleus
- ZFN causes disruption of targeted gene

1. transfer of modified embryos to foster mothers
- birth of founder animals

or

2. culture-growth of modified embryo (e.g. zebrafish)
- birth of founder animals

20

What are knockout models currently availabe?

- neurodegenerative disorders in rats
parkinson's
autism (Fmr1, Nlgn3)
schizophrenia
alzheimer's disease