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Flashcards in Lecture 25 Deck (17):

Mouse knock out:

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



- 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


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


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)


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


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


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


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


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)


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


Drosohpila complementation:

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


Mouse recombination:

- Difficult
- homologous and non-homologous



- 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


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


What is CRISPR/Cas?

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


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


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