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21 fall: Dev Bio > Lecture 7 manipulating genome > Flashcards

Lecture 7 manipulating genome Flashcards

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Biology 101 flashcards
1
Q

4 ways to clone

A

reproductive
embryonic
Therapeutic
Genetic

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2
Q

SCNT is a form of ___ cloning

A

reproductive cloning

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3
Q

iPSC is used in what type of cloning

A

therapeutic

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4
Q

what are iPSC

A

induced pluripotent stem cells

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5
Q

why do we use the mouse to clone

A

cheap
well known embryogenesis
ES cells can be cultured easily
-genetic manipulation

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6
Q

what do you need to do gene targeting

A

transgene
ability to manipulate genomic DNA- make cuts
cell line to make a modified animal

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7
Q

what do you need to consider before changing a gene

A

deletion size
delete protein domains
delete entire protein
when should the deletion happen

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8
Q

what is a transgene

A

piece of DNA that we want inserted into another piece of DNA

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9
Q

two parts of transgene

A

promoter

gene of interest

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10
Q

what are some things to consider about the promoter of a transgene

A

tissue specific
development/stage specific
constantly on?
can be turned on?

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11
Q

what are some things to consider about the gene of interest of a transgene

A

reporter gene
normal gene
mutant gene
toxin for death

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12
Q

what is an example of a reporter gene

A

GFP, LacZ, Cre

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13
Q

Pros of using transgenes

A

can express any gene from any organism

can introduce any size piece of DNA

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14
Q

Cons of using transgenes

A

piece gets randomly put into genome sequence

piece can be duplicated -100 times

piece might stop normal gene function if put in wrong spot

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15
Q

NHEJ

A

non homologous end joining

type of DSB repair

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16
Q

explain how you can use DSB to insert transgene

A

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

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17
Q

define homologous overlap

A

when transgene has start and ends pieces identical to breaks in DNA piece.

18
Q

explain how to make conditional gene knockout

A

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

19
Q

Cre recombinase works on

A

two cis LoxP sites

20
Q

explain how loxP sites work

A

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)

21
Q

why use conditional knockout

A

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
22
Q

Cre recombinase can be expressed using the drug

A

tetracycline

23
Q

3 ways to make gene cutting better

artificial restriction enzymes

A
  • Zinc-finger nuclease (ZFN)
  • Transcription activator-like effector nuclease (TALEN)
  • CRISPR/Cas9
24
Q

ZFN

A

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)

25
Q

TALEN

A

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)

26
Q

RE

A

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

27
Q

explain RE

A

restriction endonucleases recognize and cut 6-8 bp sequences.

will cut every 4^n bp

not practically because it makes too many cuts

28
Q

explain TALEN and ZFN

A

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)

29
Q

disadvantages of TALEN and ZFN

A
  • expensive
  • time consuming
  • not very effiecent maybe 1-3 protein sets will actually cut
30
Q

Explain CRISPR/Cas9

A

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

31
Q

three ways to generate clones

A

genetically modify ESC and produce chimeras

pronuclear injection of DNA into fertilized oocyte

SCNT

32
Q

explain genetically modify ESC and produce chimeras

A

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
33
Q

what is an easy way to see if chimera is born

A

blastocyst is from black mouse
ES cells are from brown mouse

brown babies higher chance of being chimera

34
Q

explain pronuclear injection of DNA into fertilized oocyte

A
  • make transgene
  • inject into fertilized oocyte
  • transfer to foster
  • birth
  • DNA analysis
  • breeding to establish transgenic lines
35
Q

explain SCNT

A

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
36
Q

why is no sperm needed in SCNT

A

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

37
Q

large animal application of transgene

A

create biopharmaceutical proteins

38
Q

large animal application of SCNT

A

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

39
Q

in what situations would a transgene randomly integrated into the genome vs a transgene inserted into a specific location of the genome be required

A

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.

40
Q

advantages and disadvantages of SCNT

A
  • expensive
  • 5% success rate
  • aging issue- telomeres same length as donated somatic cells
  • can only clone DNA not attitude

Pros:
create exact DNA copy

41
Q

advantages and disadvantages of pronuclear injection of DNA into fertilized oocyte

A

Cons:

  • transgene gets put in random spot
  • can work or not work

Pros:
***

42
Q

advantages and disadvantages of modify ESC and produce chimeras

A

Cons:
-doesn’t always work

Pros:
***

21 fall: Dev Bio (21 decks)

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