lecture 18 Flashcards
Genetic manipulation of mammalian animals and cells gene-targeting and recent advances
What is transgenesis?
the process of transferring genes to animals
What is a transgenic animal?
an animal that carries a transgene
What is a transgene?
a foreign gene
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
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
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
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
What is homologous recombination?
- an exchange between similar genetic material
What is heterologous recombination?
- an exchange between different genetic material
- random integration
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
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)
What are knockouts?
- gene-targeting (precise location)
- loss of function
- null mutant
- no functional protein
What are knockins?
- gene-targeting
- modification
- may be functional protein
- may be other species gene (protein)
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
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
