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Biol 200 > Lecture 31 - Experimental gene targeting > Flashcards

Lecture 31 - Experimental gene targeting Flashcards

1
Q

Gene targeting goal

A

Mutating or interfering with function of a specific gene

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2
Q

Gene knock-out def

A

Disruption of a specific gene

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3
Q

Gene knock-in def

A

Replacement or modification of a specific gene

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4
Q

What is the dominant negative method

A

Introduction of dominant mutant allele causing the same phenotype as ‘‘loss of function’’

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5
Q

What is RNA interference/knock down

A

Depletion of mRNA

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6
Q

What is CRISPR/Cas 9 genome editing

A

Introduction or correction of a specific mutation

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7
Q

Knock out IN YEAST: Structure of gene of interest

A

Target yeast gene with 20 nt-flanking sequence on both sides

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8
Q

Knock out IN YEAST: How disruption construct is produced (what template and what primers)

A

Use the gene that will replace the target gene as a template (ex : KanMX gene for kanamycin resistance) and PCR with primers that have the flanking sequences of target gene )

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9
Q

Knock out IN YEAST: On what is construct insertion based

A

Based on high efficiency of homologous recombination

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10
Q

Knock out IN YEAST: Step 1 (construct is ready)

A

Transform diploid yeast cell with disruption construct and expect homologous recombination between target gene and construct

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11
Q

Knock out IN YEAST: Step 2 (after homologous recombination)

A

Select for G-418 resistance (neomycin and kanamycin resistance gene)

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12
Q

Knock out IN YEAST: Step 3 (G-418 selection finished)

A

After sporulation, four haploid spores (2 have the construct) obtained. If target gene necessary, they’re nonviable

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13
Q

Gene knockout in mice step 1 (assume we have a construct)

A

Introduce DNA construct in embryonic stem (ES) cells to disrupt allele

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14
Q

Gene knockout in mice step 2 (we have isolated stem cells that contain the construct)

A

Inject stem cells in early mouse embryos ->gives chimeras

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15
Q

Gene knockout in mice step 3 (chimeras are born)

A

Mate mice to obtain heterozygous mice and then homozygous (both alleles have construct) mice

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16
Q

Gene knockout in mice : What does the construct for target gene X look like (gene X contains 3 exons)

A

Exons 1 and 3 conserved. neo (neomycin) resistance gene replaces exon 2 and ganciclovir SENSITIVITY gene (tkHSV) added beside

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17
Q

Gene knockout in mice: 2 ways the construct for gene X could insert in the DNA in the ES cells

A

Homologous recombination

Non homologous end joining

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18
Q

Gene knockout in mice: Consequence of homologous recombination and good or bad for us

A

Construct gene replaces gene X but tkHSV does not insert cause homol. recomb. -> Resistance to G-418 and ganciclovir. good for us

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19
Q

Gene knockout in mice: Consequence of non homologous and good or bad for us

A

Construct gene + tkHSV inserts somewhere nonhomol. end joining-> Resistance to G-418 and but not to ganciclovir. bad for us

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20
Q

Gene knockout in mice: Why we don’t want nonhomologous end joining to happen and what is not in our favor

A

The target gene is not replaced. Unfortunately, nonhom. end joining happens much more frequently cause mouse genome 250x bigger than yeast

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21
Q

Gene knockout in mice: Why do we use G-418 and ganciclovir

A

We select embryonic stem (ES) cells that have the construct at the place of the gene

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22
Q

Gene knockout in mice: 2 steps of ES cells selection

A

Double selection : Positive selection and negative selection

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23
Q

Gene knockout in mice: Positive selection step (why we say positive)

A

Treatment with G-418. Cells that are G-418 positive survive. ** Non recombinant cells (didn’t take up construct at all) die **

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24
Q

Gene knockout in mice: Negative selection step (why we say negative)

A

Treatment with ganciclovir. From recombinants with a random insertion and recombinants with gene-targeted insertion, only the latter survive. (Negative cause ganciclovir negative cells survive - are not sensitive)

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25
Q

Gene knockout in mice: Once we have ES cells that are (A/A - meaning brown, X+/X- - the minus meaning one gene X is knocked out), what do we do ?

A

Insert (A/A, X+/X-) ES cells in a black mouse embryo (a/a, X+/X+) and transfer embryo into a pseudopregnant female (which has absolutely no effect on experiment)

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26
Q

Gene knockout in mice: Mixed embryo obtained, what’s the next step ? (possible progeny)

A

We might get chimeric mice (brown/black and that have X+/X- and X+/X+ cells) or black mice (X+/X+)

27
Q

Gene knockout in mice: Why do we use ES cells from a brown mouse

A

To be able to differentiate mice constituted by 2 ‘‘cell-types’’ (X+/X-) from black mice that are only made of normal cells (X+/X+)

28
Q

Gene knockout in mice: Step after obtaining progeny of brown and black mice

A

Mate chimeric mice (A/A, X+/X-, a/a, X+/X+) and black mice (a/a, X+/X+)

29
Q

Gene knockout in mice: Why is it not possible to have black mice born that are (a/a, X+/X-)

A

Must take up the brown mouse ES cells to have the disrupted gene. The embryo either took up the brown cells embryo (and would automatically be chimeric and X+/X-) or not (a/a, X+/X+)

30
Q

Gene knockout in mice: What are the possible gametes produced by the chimeric mice (with respect to colour gene and target gene X)

A

A/X+, A/X-, a/X+

31
Q

Gene knockout in mice: Why is it not possible for the chimeric mouse to produce a/X- gametes

A

Because gametes in the chimeric mouse are produced by cells that are either A/A, X+/X- OR a/X+ and each type does its own meiosis

32
Q

Gene knockout in mice: What are the possible gametes produced by the black mice (with respect to colour gene and target gene X)

A

a/X+

33
Q

Gene knockout in mice: Possible progeny from chimeric and black mice mating

A

1) Brown A/a, X+/X+
2) Brown A/a, X-/X+
3) Black a/a, X+/X+

34
Q

Gene knockout in mice: Next step after obtaining progeny from chimeric and black mice

A

Screen brown progeny DNA to identify which ones are X+/X- heterozygotes (they’re all A/a heterozygotes) and mate X+/X- heterozygotes

35
Q

Gene knockout in mice: Next step after obtaining progeny from brown heterozygotes

A

Screen progeny to identify X-/X- heterozygotes -> KNOCKOUT MICE

36
Q

Gene knockout in mice: Note about the knockout mice colour (homozygote progeny of A/a, X+/X- that mated)

A

Knockout mice could be black (a/a,X-/X-) or brown (A/a, X-/X-)

37
Q

Gene knockout in mice: What can we conclude if homozygote progeny (X-/X-) of A/a, X+/X- that mated never appear and always die in the litter

A

X target gene is necessary for life

38
Q

Cell-type specific knockout in mice: Why use this technique

A

Knocking out a gene of a certain exon in it is lethal but we still want to study that gene so we express it in a particular cell-type

39
Q

Cell-type specific knockout in mice: (Say all mice have 3 exons gene named X) First type of mouse used for breeding ?

A

Mouse with exon 2 flanked by loxP sites

40
Q

Cell-type specific knockout in mice: What are loxP sites

A

Sites that are specific for DNA recombination

41
Q

Cell-type specific knockout in mice: Second type of mouse used for breeding

A

Mice that are heterozygous for gene X knockout and where all cells carry cre gene

42
Q

Cell-type specific knockout in mice: What is the cre gene and what is particular about its promoter

A

Cre is a recombinase gene from bacteriophage and its promoter is cell-type specific (only expressed in muscle cells)

43
Q

Cell-type specific knockout in mice: X gene and cre gene in cells of progeny of first 2 mice

A

All cells have a copy of the lox-P modified gene X, a copy of gene X knockout and cre gene

44
Q

Cell-type specific knockout in mice: What happens in cells that do not express Cre (not muscle)

A

X gene accomplishes its functions normally (3 exons obtained, etc.)

45
Q

Cell-type specific knockout in mice: What happens in cells that express cre (muscle cells)

A

Cre directs recombination between the two LoxP regions and gene X only has exons 1 and 3 -> Function disrupted

46
Q

Gene fct inactivation by dominant negative allele: Goal

A

Use it when members of a gene family are redundant and do the same thing but we still want to study that gene -> Multiple knockout = too laborious

47
Q

Gene fct inactivation by dominant negative allele: Principle (what the protein we express with our construct looks like + effect on its function)

A

Make a mutant of a gene where a truncated protein is expressed to obtain part of its function

48
Q

Gene fct inactivation by dominant negative allele: Example

A

Changing a GTPase to make it unable to leave GEF after its bound to it so that all other GTPases can’t be activated (all gene family blocked)

49
Q

Knock down by RNA interference: Goal

A

Degrade mRNA of a specific gene

50
Q

Knock down by RNA interference: Principle

A

Produce dsRNA in vitro and insert into cell. Will be cut into fragments by Dicer and what will form fragments complementary to the target mRNA

51
Q

Knock down by RNA interference: Where dsRNA fragments cut by Dicer go and consequence

A

Go in the RISC complex, will hybridize w/ the mRNA and induce endonuclease activity of RISC (RNA-induced silencing complex)

52
Q

Knock down by RNA interference: Which RNA size can be injected in the cell and which size can be efficiently transfected

A 
Long dsRNA can be microinjected but no efficiently transfected
Short dsRNA (short interfering, siRNA) can be transfected
53
Q

Knock down by RNA interference: How dsRNA can be produced in vitro

A

Introduce cDNA of the target gene in a plasmid and transcribe it. Repeat but by putting it in the other direction in the plasmid. Complementary RNA strands will make dsRNA

54
Q

Knock down by RNA interference: What is required for production of the dsDNA in vitro

A

A strong promoter, RNA Pol, rNTPs

55
Q

Knock down by RNA interference: How to produce in vivo dsRNA

A

Introduce insert in a plasmid’s that will form shRNA (small or short hairpin RNA and that is complementary to target gene/its mRNA) -> Dicer cuts it, etc.

56
Q

Knock down by RNA interference: What is required for production of the dsRNA in vivo

A

Promoter on plasmid.

57
Q

CRISPR-Cas9 system: Take away message

A

is an RNA-guided endonuclease

58
Q

CRISPR-Cas9 system: What happens naturally (steps before until Cas9)

A

In infected bacteria, bacteria produces RNA complementary to the virus and this RNA binds to Cas9

59
Q

CRISPR-Cas9 system: What is Cas9

A

endonuclease

60
Q

CRISPR-Cas9 system: What normally happens in virally infected bacteria after Cas9 is bound to the copied RNA

A

Cas9 looks for DNA complementary to the RNA it carries and cuts in the compl. DNA sequence when finds it

61
Q

CRISPR-Cas9 system: What normally happens after a dsDNA break (like after Cas9 cutting) (hint : as usual)

A

Homologous recombination based on other copy of the chromosome (paternal or maternal) or nonhomologous end joining

62
Q

CRISPR-Cas9 system: What CRISPR-Cas9 system does to introduce mutations

A

Makes sure the broken strand uses an HDR (homology directed repair template) that we constructed

63
Q

CRISPR-Cas9 system: Mutations in NHEJ vs with HDR

A

NHEJ: Disruptions by small insertions or deletions
HDR: Corrections or insertions

Biol 200 (30 decks)

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