Lecture 25

Question 1

Mouse knock out:

Answer 1

• Inactivates gene function by inserting the selectable marker inside the gene of interest
• Using NeoR and gene of interest

Question 2

Knock-in:

Answer 2

• HSV-TK and neoR as a selectable marker, flanked with the gene of interest you are trying to introduce.
• Use of NeoR to select for transformants
• Native promoter, genomic location but introduces flanking vector sequences inactivating the genomic copy

Question 3

Double replacement (not the yeast-two-step gene replacement!):

Answer 3

1. Introduce a selectable marker into the genome

2. Replace the selectable marker with the specific mutation you wish to introduce into the genome

Question 4

Double replacement, step 1:

Answer 4

• Introduce hprt+ into hprt- ES cells at the required site
• This will occur through homologous recombination
• Select for HPRT+ (encodes an enzyme for purine synthesis)
• Use HAT medium (hprt- cells can’t survive as there is no purine, so they require HPRT+ for purine synthesis)

Question 5

Double replacement, step 2:

Answer 5

• Introduce mutated sequences
• Select in 2-thioguanine (2-TG) medium, to keep only hprt- cells
• Homologous recombination allows replacement
• The mutated genomic copy will be left, with no other sequences around it

Question 6

Other strategies developed for ‘clean’ mutation of genomic sequence, eg) Cre/loxP system:

Answer 6

• Cre: 38kDa recombinase from bacteriophage P1
• Catalyses site specific loxP sites
• Can be used to remove genomic sequences and introduce loxP sites
• introduce loxP sites by positive-negative selection
• End up with a floxed allele

Question 7

Cre/loxP, action of Cre recombinase

Answer 7

• Introduce Cre recombinase into cells carrying the ‘floxed’ allele
• Cre recombinase removes sequences flanked by loxP sites, and deletion of sequences follows

Question 8

Using Cre/lox to introduce specific mutations:

Answer 8

Introduce DNA carrying a ‘floxed’ selectable marker and the mutation of interest

• Then use Cre recombinase to remove the neoR marker
• Finish with the genomic copy with the mutation with the loxP site in an intron (so that it doesn’t interfere with the action of the gene of interest

Question 9

Cre recombinase expression can be regulated:

Answer 9

• Drive Cre expression at specific times of development or in specific tissues
• Generate a mouse line that carries a ‘floxed’ allele
• LoxP sites in introns will have no effect on the genes and no effect on the mouse
• Generate a mouse line expressing Cre recombinase in specific tissues or developmental stages
• This can be introduced into the mouse, and will have no effect on the mouse (unless there are loxP sites)

Question 10

Crossing the lines of mice will result in:

Answer 10

• Cross: Expressed Cre in specific tissue
• With: Contains ‘floxed’ gene in all tissues
• Activation of Cre expression in specific tissue so loss of function in specific tissues occurs

Question 11

Drosohpila complementation:

Answer 11

• Not easy
• Low frequency
• Non-homologous
• Integration via P-element

Question 12

Mouse recombination:

Answer 12

• Difficult

- homologous and non-homologous

Question 13

CRISPR/Cas:

Answer 13

• Allows you design a mutation in any organism, which can be knock-out or other sorts of mutations
• Not dependent on P-elements or Ti-plasmids
• Based on DNA being introduced with homology to the target sequences

Question 14

How was CRISP discovered?

Answer 14

• CRISPR uses numerous direct repeats separated by variable sequences (spaces) and adjacent to the Cas genes
• Genomic sequcneing revealed that the spacer regions correspond to viral and plasmid sequences
• Recognised as a microbial adaptive immune system, a record of all previously encountered pathogens

Question 15

What is CRISPR/Cas?

Answer 15

• A large family of proteins that have funciton domains including nucleases, polymerases and helicases, including Cas9

Question 16

What is Cas9?

Answer 16

• A large monomeric DNA nuclease
• Guided to a DNA target sequence by a complex of two non-coding RNAs: crRNA and tracerRNA
• Cas9 + sgRNA (hybrid RNA) = CRISPR

Question 17

How is CRISPR used?

Answer 17

• Cas9 and sgRNA form a complex
• Cas9 can but both strands of DNA and the cut can then be repaired by DNA repair systems
• The sgRNA sequence determines the location of the cuts in the DNA made by Cas9
• This is targeted mutation of the genome with the possiblilty of creating multiple mutations at any time