RR16: Molecular Biology of Gene Targeting

Question 1

what is the best way to figure out the function of a gene?

Answer 1

test what happens to cell/organism when you remove that gene function

Question 2

how do you obtain mutants?

Answer 2

by randomly introducing mutations or mutagens and collecting mutants corresponding to different phenotype

Question 3

what is forward genetic analysis?

Answer 3

you’re looking for mutants that have a phenotype but you dont know what the gene is that corresponds too that phenotype

Question 4

what is reverse genetic analysis and an example? and what is it also referred to

Answer 4

remove that gene function and analyse the phenotypes that arise once you remove the gene you’re interested in, disrupt gene and then assess its function
RNAi
gene targeting

Question 5

what can embryonic stem cells do?

Answer 5

homologous recombination

Question 6

what is the difference in terms of the flanking sequence for yeast vs mouse?

Answer 6

you need to know a lot more of the flanking sequence in mice, need to know more about the gene already

Question 7

how can you modify that gene in mice?

Answer 7

• Call it a replacement/disruption construct, flanking sequence of a dominant selectable marker (neomycin resistance)
• Another gene included, outside of the region of homology called thymine kinase that will metabolize a drug to make it super toxic, and cells cant live
• You do this to direct the homologous recombination event and not random insertion event
• transfert that into ES cells and elect using the drug (neomycin or G-418), select for the cells where that construct was integrated
• cells that did homologous recombination or non homologous recombination (random insertion), latter present a lot more
• when you have the homologous recombination, you will not have the thymine kinase gene, because it was outside those regions of homology
• in random insertion, thymine kinase will be inserted as well
• select against the presence of thymine kinase by putting those cells on the antiviral drug ganciclovir
• In the presence of thymidine kinase it will kill the cells
• so the cells that took up DNA randomly will be eliminated and you will only have the ones that did homologous recombination left
• first round of selection: select for integration of construct second selection: select for ES cells that did homologous recombination

Question 8

what happens after you have successfully modified those mouse cells?

Answer 8

• when you put these cells in an embryo and in a proper context, they can give rise to a proper functioning mouse
• You inject them into a blastocyst
• ES cells came from a mouse with a brown coat color (dominant)
• Inject them into a blastoderm that has black coat color (recessive)
• The cells mix and divide and contribute to the various tissues
• Put into a pseudo pregnant mouse
• It grows till the pups come out, which can come in two types: black (unaffected by you injecting those cells)
• Tiger like; brown and black, because you put in those brown coat color cells, that is the animal that was engineered

Question 9

how can you get a homozygous animal that has been manipuated for that gene segment?

Answer 9

• You hope that the cells affected are germ cells

Question 10

what are the two situations after the animal is born?

Answer 10

• Very often once they become homozygous they die and you have to figure out why
• You can find out that the animals are fine: other gene products compensate for the loss, its redundant
• You don’t find out much more about gene function

Question 11

how can you make transgenic mice?

Answer 11

• make it by doing transgenic mice
• Make a transcriptional fusion, introduce it into an embryo, and see where the gene is expressed
• You can take gene products and make them into dominant/negative gene products by removing the DNA binding domain
• Introduce it transgenically into a mouse and suddenly you can see phenotypes that are typical of loss of functions
• you can also do RNAi
• what you have to do is make an expression construct, inject it into one of the two pronuclei at the point of fertilization of an ovocyte
• Transfer injected eggs into pseudopregnant mom and allow her to generate her progeny
• high % will have integrated that DNA into chromosome
• Breed them to have a stably integrated trans gene in a mouse strain

Question 12

what does CRISPR stand for?

Answer 12

Clustered regularly interspaced short palindromic regions

Question 13

what do those CRISPR regions give when transcribed?

Answer 13

transcribed in the bacteria to make a primary transcript that has the regions and RNAs that correspond to those bacteriophae genes (CRISPR RNAs)

Question 14

what does CRISPR RNA do?

Answer 14

• Trans activating CRISPR RNA that interact complimentarity with those interspaced repeated
• It will help to mature that primary transcript into individual crRNAs

Question 15

what is crRNA recognised by?

Answer 15

a protein in the bacteria called CAS9
RNA binding protein that interacts with that stem loop

Question 16

how will CAS9 use crRNA to affect its target?

Answer 16

• It will use the crRNA to take it to a target DNA on an invading bacteriophage
• When the crRNA recognizes its complimentary sequences, CAS9 will carry out a double stranded nucleolytic cleavage of that DNA: immune response to the bacteriophage

Question 17

what are the two endonucleases on CAS9?

Answer 17

HNH and a rubC
* One specific for one strand the other specific for the other strand of the DNA

Question 18

how can CRISPR CAS9 be used to modify/edit the genome?

Answer 18

• you need to introduce CAS9 and the guide RNA into the reaction
• Need at least two different trans genes
• Newly optimized cas (have NLS sequence and you have drive it with the promoters that correspond to it
• Once they come together they sit down and catalyse that break
• Cell recognizes the break right away and does not do a good job at fixing it
• Will use non homologous end joining to repair the break but its not correct and will not last very long
• you end up getting deletions and insertions and nonsense mutations
• Very efficient way of getting mutations in a gene that you are interested in, get a whole family of indels (insertions and deletions)
• You will disrupt a reading frame and get an incorrectl made protein
• if you can introduce a copy of DNA into those cells again (wild type or modified) as long as there are homologous sequences that the cell can recognise you can repair that break, and by doing that you introduce that piece of DNA
• Alter a protein very quickly by introducing mutations in that repair trans gene
• very powerful: correcting and editing the genome

Question 19

how can you generate an RNA such that you could eliminate the necessity of a trans activating crRNA?

Answer 19

• you can make a single RNA that has the same kinds of stem loops and add on an RNA sequence that recognizes almost any DNA target and any substrate that you want to eliminate
• Single guide RNA: stem loops that is recognized by CAS9 + any sequence that you want to introduce to target the cleavage of some DNA that you want to cut

Question 20

what does the specificity conferred by the sequence rely on?

Answer 20

protospacer adjacent motif (PAM motif)
* For the cleavage to work, guide RNA has to be engineered to base pair with sequences next to this PAM motif (trinucleotide sequence), which could be any nucleotide followed by GG, 3’ to the site that you’re trying to hit
and then you get the two cleavages with the two endonucleases