Lecture 31 - Experimental gene targeting Flashcards
Gene targeting goal
Mutating or interfering with function of a specific gene
Gene knock-out def
Disruption of a specific gene
Gene knock-in def
Replacement or modification of a specific gene
What is the dominant negative method
Introduction of dominant mutant allele causing the same phenotype as ‘‘loss of function’’
What is RNA interference/knock down
Depletion of mRNA
What is CRISPR/Cas 9 genome editing
Introduction or correction of a specific mutation
Knock out IN YEAST: Structure of gene of interest
Target yeast gene with 20 nt-flanking sequence on both sides
Knock out IN YEAST: How disruption construct is produced (what template and what primers)
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 )
Knock out IN YEAST: On what is construct insertion based
Based on high efficiency of homologous recombination
Knock out IN YEAST: Step 1 (construct is ready)
Transform diploid yeast cell with disruption construct and expect homologous recombination between target gene and construct
Knock out IN YEAST: Step 2 (after homologous recombination)
Select for G-418 resistance (neomycin and kanamycin resistance gene)
Knock out IN YEAST: Step 3 (G-418 selection finished)
After sporulation, four haploid spores (2 have the construct) obtained. If target gene necessary, they’re nonviable
Gene knockout in mice step 1 (assume we have a construct)
Introduce DNA construct in embryonic stem (ES) cells to disrupt allele
Gene knockout in mice step 2 (we have isolated stem cells that contain the construct)
Inject stem cells in early mouse embryos ->gives chimeras
Gene knockout in mice step 3 (chimeras are born)
Mate mice to obtain heterozygous mice and then homozygous (both alleles have construct) mice
Gene knockout in mice : What does the construct for target gene X look like (gene X contains 3 exons)
Exons 1 and 3 conserved. neo (neomycin) resistance gene replaces exon 2 and ganciclovir SENSITIVITY gene (tkHSV) added beside
Gene knockout in mice: 2 ways the construct for gene X could insert in the DNA in the ES cells
Homologous recombination
Non homologous end joining
Gene knockout in mice: Consequence of homologous recombination and good or bad for us
Construct gene replaces gene X but tkHSV does not insert cause homol. recomb. -> Resistance to G-418 and ganciclovir. good for us
Gene knockout in mice: Consequence of non homologous and good or bad for us
Construct gene + tkHSV inserts somewhere nonhomol. end joining-> Resistance to G-418 and but not to ganciclovir. bad for us
Gene knockout in mice: Why we don’t want nonhomologous end joining to happen and what is not in our favor
The target gene is not replaced. Unfortunately, nonhom. end joining happens much more frequently cause mouse genome 250x bigger than yeast
Gene knockout in mice: Why do we use G-418 and ganciclovir
We select embryonic stem (ES) cells that have the construct at the place of the gene
Gene knockout in mice: 2 steps of ES cells selection
Double selection : Positive selection and negative selection
Gene knockout in mice: Positive selection step (why we say positive)
Treatment with G-418. Cells that are G-418 positive survive. ** Non recombinant cells (didn’t take up construct at all) die **
Gene knockout in mice: Negative selection step (why we say negative)
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)
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 ?
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)
Gene knockout in mice: Mixed embryo obtained, what’s the next step ? (possible progeny)
We might get chimeric mice (brown/black and that have X+/X- and X+/X+ cells) or black mice (X+/X+)
Gene knockout in mice: Why do we use ES cells from a brown mouse
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+)
Gene knockout in mice: Step after obtaining progeny of brown and black mice
Mate chimeric mice (A/A, X+/X-, a/a, X+/X+) and black mice (a/a, X+/X+)
Gene knockout in mice: Why is it not possible to have black mice born that are (a/a, X+/X-)
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+)
Gene knockout in mice: What are the possible gametes produced by the chimeric mice (with respect to colour gene and target gene X)
A/X+, A/X-, a/X+
Gene knockout in mice: Why is it not possible for the chimeric mouse to produce a/X- gametes
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
Gene knockout in mice: What are the possible gametes produced by the black mice (with respect to colour gene and target gene X)
a/X+
Gene knockout in mice: Possible progeny from chimeric and black mice mating
1) Brown A/a, X+/X+
2) Brown A/a, X-/X+
3) Black a/a, X+/X+
Gene knockout in mice: Next step after obtaining progeny from chimeric and black mice
Screen brown progeny DNA to identify which ones are X+/X- heterozygotes (they’re all A/a heterozygotes) and mate X+/X- heterozygotes
Gene knockout in mice: Next step after obtaining progeny from brown heterozygotes
Screen progeny to identify X-/X- heterozygotes -> KNOCKOUT MICE
Gene knockout in mice: Note about the knockout mice colour (homozygote progeny of A/a, X+/X- that mated)
Knockout mice could be black (a/a,X-/X-) or brown (A/a, X-/X-)
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
X target gene is necessary for life
Cell-type specific knockout in mice: Why use this technique
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
Cell-type specific knockout in mice: (Say all mice have 3 exons gene named X) First type of mouse used for breeding ?
Mouse with exon 2 flanked by loxP sites
Cell-type specific knockout in mice: What are loxP sites
Sites that are specific for DNA recombination
Cell-type specific knockout in mice: Second type of mouse used for breeding
Mice that are heterozygous for gene X knockout and where all cells carry cre gene
Cell-type specific knockout in mice: What is the cre gene and what is particular about its promoter
Cre is a recombinase gene from bacteriophage and its promoter is cell-type specific (only expressed in muscle cells)
Cell-type specific knockout in mice: X gene and cre gene in cells of progeny of first 2 mice
All cells have a copy of the lox-P modified gene X, a copy of gene X knockout and cre gene
Cell-type specific knockout in mice: What happens in cells that do not express Cre (not muscle)
X gene accomplishes its functions normally (3 exons obtained, etc.)
Cell-type specific knockout in mice: What happens in cells that express cre (muscle cells)
Cre directs recombination between the two LoxP regions and gene X only has exons 1 and 3 -> Function disrupted
Gene fct inactivation by dominant negative allele: Goal
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
Gene fct inactivation by dominant negative allele: Principle (what the protein we express with our construct looks like + effect on its function)
Make a mutant of a gene where a truncated protein is expressed to obtain part of its function
Gene fct inactivation by dominant negative allele: Example
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)
Knock down by RNA interference: Goal
Degrade mRNA of a specific gene
Knock down by RNA interference: Principle
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
Knock down by RNA interference: Where dsRNA fragments cut by Dicer go and consequence
Go in the RISC complex, will hybridize w/ the mRNA and induce endonuclease activity of RISC (RNA-induced silencing complex)
Knock down by RNA interference: Which RNA size can be injected in the cell and which size can be efficiently transfected
Long dsRNA can be microinjected but no efficiently transfected Short dsRNA (short interfering, siRNA) can be transfected
Knock down by RNA interference: How dsRNA can be produced in vitro
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
Knock down by RNA interference: What is required for production of the dsDNA in vitro
A strong promoter, RNA Pol, rNTPs
Knock down by RNA interference: How to produce in vivo dsRNA
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.
Knock down by RNA interference: What is required for production of the dsRNA in vivo
Promoter on plasmid.
CRISPR-Cas9 system: Take away message
is an RNA-guided endonuclease
CRISPR-Cas9 system: What happens naturally (steps before until Cas9)
In infected bacteria, bacteria produces RNA complementary to the virus and this RNA binds to Cas9
CRISPR-Cas9 system: What is Cas9
endonuclease
CRISPR-Cas9 system: What normally happens in virally infected bacteria after Cas9 is bound to the copied RNA
Cas9 looks for DNA complementary to the RNA it carries and cuts in the compl. DNA sequence when finds it
CRISPR-Cas9 system: What normally happens after a dsDNA break (like after Cas9 cutting) (hint : as usual)
Homologous recombination based on other copy of the chromosome (paternal or maternal) or nonhomologous end joining
CRISPR-Cas9 system: What CRISPR-Cas9 system does to introduce mutations
Makes sure the broken strand uses an HDR (homology directed repair template) that we constructed
CRISPR-Cas9 system: Mutations in NHEJ vs with HDR
NHEJ: Disruptions by small insertions or deletions
HDR: Corrections or insertions
