Lecture 25 Flashcards Preview

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Flashcards in Lecture 25 Deck (17):
1

Mouse knock out:

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

2

Knock-in:

- 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

3

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

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

4

Double replacement, step 1:

- 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)

5

Double replacement, step 2:

- 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

6

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

- 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

7

Cre/loxP, action of Cre recombinase

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

8

Using Cre/lox to introduce specific mutations:

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

9

Cre recombinase expression can be regulated:

- 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)

10

Crossing the lines of mice will result in:

- 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

11

Drosohpila complementation:

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

12

Mouse recombination:

- Difficult
- homologous and non-homologous

13

CRISPR/Cas:

- 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

14

How was CRISP discovered?

- 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

15

What is CRISPR/Cas?

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

16

What is Cas9?

- 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

17

How is CRISPR used?

- 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