Lecture 7 manipulating genome Flashcards

Question

TALEN

Answer 1

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)

Answer 2

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

Answer 3

restriction endonucleases recognize and cut 6-8 bp sequences. will cut every 4^n bp not practically because it makes too many cuts

Answer 4

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)

Answer 5

- expensive - time consuming - not very effiecent maybe 1-3 protein sets will actually cut

Answer 6

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

Answer 7

genetically modify ESC and produce chimeras pronuclear injection of DNA into fertilized oocyte SCNT

Answer 8

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

Answer 9

blastocyst is from black mouse ES cells are from brown mouse brown babies higher chance of being chimera

Answer 10

- make transgene - inject into fertilized oocyte - transfer to foster - birth - DNA analysis - breeding to establish transgenic lines

Answer 11

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

Answer 12

somatic cell nucleus already diploid- already has male and female genetic information

Answer 13

create biopharmaceutical proteins

Answer 14

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

Answer 15

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.

Answer 16

- expensive - 5% success rate - aging issue- telomeres same length as donated somatic cells - can only clone DNA not attitude Pros: create exact DNA copy

Answer 17

Cons: - transgene gets put in random spot - can work or not work Pros: ***

Answer 18

Cons: -doesn't always work Pros: ***