Lecture 7 manipulating genome Flashcards
4 ways to clone
reproductive
embryonic
Therapeutic
Genetic
SCNT is a form of ___ cloning
reproductive cloning
iPSC is used in what type of cloning
therapeutic
what are iPSC
induced pluripotent stem cells
why do we use the mouse to clone
cheap
well known embryogenesis
ES cells can be cultured easily
-genetic manipulation
what do you need to do gene targeting
transgene
ability to manipulate genomic DNA- make cuts
cell line to make a modified animal
what do you need to consider before changing a gene
deletion size
delete protein domains
delete entire protein
when should the deletion happen
what is a transgene
piece of DNA that we want inserted into another piece of DNA
two parts of transgene
promoter
gene of interest
what are some things to consider about the promoter of a transgene
tissue specific
development/stage specific
constantly on?
can be turned on?
what are some things to consider about the gene of interest of a transgene
reporter gene
normal gene
mutant gene
toxin for death
what is an example of a reporter gene
GFP, LacZ, Cre
Pros of using transgenes
can express any gene from any organism
can introduce any size piece of DNA
Cons of using transgenes
piece gets randomly put into genome sequence
piece can be duplicated -100 times
piece might stop normal gene function if put in wrong spot
NHEJ
non homologous end joining
type of DSB repair
explain how you can use DSB to insert transgene
make transgene have start and ends pieces identical to breaks in DNA piece.
NHEJ will think transgene is how to fix it and put it in the middle
define homologous overlap
when transgene has start and ends pieces identical to breaks in DNA piece.
explain how to make conditional gene knockout
transgene has identical start and ends, but in the middle now contains two LoxP surrounding the gene that wants to be cut out when Cre recombinase is expressed
NHEJ will place transgene into DSB
Cre recombinase works on
two cis LoxP sites
explain how loxP sites work
loxP are 34 bp long
Cre recombinase recognizes two loxP sites and will cut the genome at those two sites
the remaining part of the genome will get put together
part of genome cut out gets tied together (makes circle)
why use conditional knockout
want to study gene function later in development
- want to disrupt gene function in specific cells or tissues or stage
- Cre recombinase can be expressed using tetracycline
Cre recombinase can be expressed using the drug
tetracycline
3 ways to make gene cutting better
artificial restriction enzymes
- Zinc-finger nuclease (ZFN)
- Transcription activator-like effector nuclease (TALEN)
- CRISPR/Cas9
ZFN
finc-finger nuclease
(type of artificial restriction enzyme that is used to increase homologous recombination- used to make cuts in genome so transgenes can be added)
TALEN
transcription activator-like effector nuclease
((type of artificial restriction enzyme that is used to increase homologous recombination- used to make cuts in genome so transgenes can be added)
RE
restriction endonucleases
increases efficiency of Homologous recombinations (cuts in genome)
cuts every 4^6-8 bp
not used because it would make too many cuts
explain RE
restriction endonucleases recognize and cut 6-8 bp sequences.
will cut every 4^n bp
not practically because it makes too many cuts
explain TALEN and ZFN
Zinc-finger nuclease (ZFN)
-Transcription activator-like effector nuclease (TALEN)
recognize long stretched of bases within the genome and makes DSBs
uses cleavage domain (Fok1)
disadvantages of TALEN and ZFN
- expensive
- time consuming
- not very effiecent maybe 1-3 protein sets will actually cut
Explain CRISPR/Cas9
RNA guided platform to make cuts in genome at specific spots
- short “guide RNA” 20 bp target loci for directing cas9 nuclease.
- cas9 cleavage is repaired by either NHEJ or HDR in tandem with a donor
very effective
three ways to generate clones
genetically modify ESC and produce chimeras
pronuclear injection of DNA into fertilized oocyte
SCNT
explain genetically modify ESC and produce chimeras
alter embryonic stem cells
- inject mutated ES cells into blastocyst at E3.5
- transfer to foster mother
- birth of chimera
- breed chimeras to obtain homozygotes
- analysis of phenotype
what is an easy way to see if chimera is born
blastocyst is from black mouse
ES cells are from brown mouse
brown babies higher chance of being chimera
explain pronuclear injection of DNA into fertilized oocyte
- make transgene
- inject into fertilized oocyte
- transfer to foster
- birth
- DNA analysis
- breeding to establish transgenic lines
explain SCNT
somatic cell nuclear transfer
- somatic cell taken from patient and nucleus taken out
- donor egg harvested
- nucleus taken out of egg
- somatic nucleus put into ‘empty’ egg
- embryo formed
- transfered to surrogate
- clone is born
why is no sperm needed in SCNT
somatic cell nucleus already diploid- already has male and female genetic information
large animal application of transgene
create biopharmaceutical proteins
large animal application of SCNT
increase animal quality- a specific animal tastes really good or has good muscles or is immune for something- we make clones of that specific animal to keep the good stuff coming
in what situations would a transgene randomly integrated into the genome vs a transgene inserted into a specific location of the genome be required
depends on animal
if you don’t know whole sequence use random,
-GFP can be random, doesn’t matter where it goes just expresses itself
if you know sequence of genome and you want to express a transgene at a specific location you can use Crispr to cut at right place.
So the situation depends on the animal that you are interested in targeting for manipulation. Some animals have their genomes sequenced, so you can look up the sequences for designing homology arms, for targeting a transgene to a specific location. However, some species don’t have their genome sequenced..or maybe there’s just a partial sequence; so this situation will require targeting a transgene into a random place in the genome. In addition, if you don’t know the sequence of the gene/region you are targeting, you can’t design CRISPR oligos for introducing DSBs into a particular region. An example would be if you wanted to express GFP in a species of alpacas (like the vicuna alpaca) - you would need to use the random integration approach.
However, if you know the sequence of the animal that you want to manipulate (for example, a black6 mouse), then you could use a specific targeting strategy to insert the transgene into a specific region of the region. An example would be to insert GFP into mice, into the region of the noncoding RNA Xite on the X chromosome. This would require designing CRISPR oligos for introducing DSBs at the Xite locus, and then using a transgene with homology arms for the Xite region.
advantages and disadvantages of SCNT
- expensive
- 5% success rate
- aging issue- telomeres same length as donated somatic cells
- can only clone DNA not attitude
Pros:
create exact DNA copy
advantages and disadvantages of pronuclear injection of DNA into fertilized oocyte
Cons:
- transgene gets put in random spot
- can work or not work
Pros:
***
advantages and disadvantages of modify ESC and produce chimeras
Cons:
-doesn’t always work
Pros:
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