Cloning, Screening and Manipulation

Question 1

General strategy of yeast two-hybrid

Answer 1

1. Protein X expressed as a hybrid with DNA binding domain of a TF for ‘bait’
2. Library of prey generated -> each clone expressed as a hybrid protein with TF transactivation domain
3. Final component = reporter gene activated by 2-hybrid TF
4. Matinb between opposite yeast cells -> diploid cells carrying both components
5. reporter gene activated in cells where components interact -> isolation + identification

Question 2

A yeast-two hybrid is

Answer 2

an in vivo interaction screening method

• > protein interactions assayed in vivo
• > confirm interactions between known proteins + screen for unkown proteins that interact with a given protein of interest

Question 3

TF’s generally contain:

Answer 3

1. Individual binding domain

2. Transactivation domain

Question 4

Expression cloning

Answer 4

expression vectors used to generate a cDNA library with each clone expressing 1 protein

Question 5

N = ln (1-p)/ ln(1-f) what do the variables stand for and what is the caveat of using this equation

Answer 5

N = number of unique clones
p = probability sequence is present at least once
f = fractional size

-> assume each fragment clones equally well

Question 6

The difference between manipulation of genome in animals and humans is that

Answer 6

in humans gemrline

Question 7

Random integraion

Answer 7

-> often as multiple clones
Small percentage of cells integrate added DNA into genome, added DNA has a selection marker
-> establish clonal cell lines + efficiency
- 1-2 orders lower than with transient expression

Question 8

Random integration in mice

Answer 8

Locus is not equally distributed through genome in mice

-> DNA is injected into fertilised oocytes + re-implanted into foster pseudo pregnant female mice -> germline

Question 9

Problems with random integration in transgenic mice

Answer 9

1. Often dissapointing expression in CNS

2. Longer DNA better e.g. BAC

Question 10

Targeting vectors promote _____ by ____ when

Answer 10

Homologous recombination, inclusion of homology region
-> homologous to target gene so targeting vector can synapse with edogenous DNA
, introduced into ES cells

Question 11

To produce knockout mice:

Answer 11

Endogenous loci disrupted

Question 12

To increase efficency of homologous recombination

Answer 12

If linearised before insertion

Question 13

Site-specific recombination

Answer 13

Allows precise manipulation of genomes in organisms where gene targetting is inefficient
-> delete unwanted transgenes

Question 14

An example of site-specific recombination

Answer 14

Cre/lox system from P1 phage :

- Cre recombinase combines lox sequences ( short target sequences )

Question 15

Guide RNA

Answer 15

A guide RNA directs an engineered nuclease to act at locations defined by the seqeunce of guide RNA.