Transgenic and gene targeting technologies

Question 1

Identifying a gene of interest

Answer 1

> transcriptome profling
protein-based methods
whole genome sequencing

Question 2

Transcriptome profiling

Answer 2

Look at expression of RNA in particular cell type
e.g. cancer cell line

> RT-PCR
DNA microarrays
RNA-seq

Question 3

RNA sequencing

Answer 3

1. turn all RNA into DNA
2. sequence using next gen. sequencing
3. show you genes up or down regulated

Question 4

Protein-based methods

Answer 4

> 1- and 2-hybrid screening

> Immunoprecipitation

Question 5

Immunoprecipitation

+ mass spectrometry

Answer 5

Precipitating a protein antigen out of solution
- using an antibody that specifically binds to that protein

MS = separates proteins based on mass and charge

Question 6

Whole genome sequencing

Answer 6

GWAS

= genome-wide association studies

Question 7

In vitro alternatives to in vivo methods for studying gene function

Answer 7

> cell + 2D tissue culture

> 3D tissue culture + organoids

Question 8

In vitro limitations compared to in vivo

Answer 8

> not all cell types amenable to in vitro
limited matrix + cell interactions
limited interstitial, endocrine + other factors
no recapitulation of forces
no developmental modelling
no organism-level analysis e.g. response to stress

Question 9

Manipulating genes in vivo

Answer 9

Usually involves manipulation in bacteria

Transgenesis

Conventional gene targeting

Genome editing

Question 10

Transgenesis

What does this enable?

Answer 10

process of introducing a gene from 1 org into another

Visualisation of gene expression in whole animal

Question 11

Transgenesis

- process

Answer 11

1. Inject tg DNA into 1-cell embryo
   - pronuclear injection
2. Tg integrates into genome in quasi-random fashion
3. Transfer embryo into mother
4. Embryo -> offspring

(all cells in offspring have tg)

Question 12

Transgenesis efficiency

Answer 12

approx 10% of offspring = transgenic

Question 13

Gene targeting: ES cells

Answer 13

= targets particular region in genome

ES cells derived from ICM of blastocyst
- can contribute to blastocyst development if injected

Question 14

What does gene target support?

Answer 14

High rates of homologous recombination

Question 15

ES cell gene targeting method

Answer 15

1. make targeting vector DNA construct
2. • selection
     - select with G418
     OR - selection with Ganciclovir
3. use reporter e.g. GFP

Question 16

When to use + or - selection for ES cell targeting?

Answer 16

+ for all TV integration events

• for homologous integration events

Question 17

Targeting vector

- homology arms

Answer 17

Each side of targeted insertion

- recombine by Homology Directed Repair with genome

Question 18

From mutant ES cells to mutant mice

Answer 18

1. Inject 8-10 targeted ES cells into blastocyst
2. Transfer blastocyst into pseudo mother
3. Blastocyst develops + ES cells integrate into development
4. Offspring contain cells from embryo + targeted ES cells (revealed by coat colour)

Question 19

Example of gene targeting

Answer 19

Vomaronasal (pheromone) receptor 2

lacZ makes bacteria blue
- blue-white selection

Question 20

How do the ES cells affect the next generation of mice?

Answer 20

ES cells contribute to all germ layers + sometime germline
-> next gen homozygous for ES cell
= mutation

Question 21

Gene editing

Answer 21

DNA is inserted, deleted, modified or replaced in the genome

Question 22

Gene editing

- method

Answer 22

1. Create ds break at specific place in genome
   - via ZFNs or TALEN
2. cell machinery repairs dsb
   - results in NHEJ or HDR

Question 23

ZFN

Answer 23

Zinc Finger Nuclease
- come from mammalian TFs

Tandem ZnFs fused to Fok1 nuclease

Question 24

ZFN and Fok1 nuclease

Answer 24

1. Remove Fok1 recognition domain + replace with ZFNs
2. Fok1 only works as dimer
3. 2 x Fok1 come together + cut ds DNA

Question 25

TALEN

Answer 25

Transcription Activator-Like Effector Nuclease Tandem TALE repeats fused to Fok1

Question 26

NHEJ

Answer 26

= non-homologous end joining (cut + paste rejoins ends) - imprecise repair - causes small insertion or deletion

Question 27

HDR

Answer 27

= Homology-direct repair (different DNA molecule used as repair template) - precise reapir

Question 28

Problem with ZFNs

Answer 28

For every mutation + cut | -> have to make left and right ZFNs

Question 29

Cas9 | - protein type + basic role

Answer 29

= RNA-guided endonuclease - helicase simple to use (scissors)

Question 30

Cas9 system components

Answer 30

> CRISPR RNA > gRNA = guide RNA > Cas9 = CRISPR-associated helicase nuclease

Question 31

CRISPR | - stands for + basic role

Answer 31

= Clustered Regularly Interspaced Short Palindromic Repeat | guides the scissors

Question 32

CRISPR-Cas9 | - mechanism

Answer 32

gRNA interacts with Cas9 | = forms a target sequence

Question 33

Benefit of CRISPR-Cas9

Answer 33

1 size fits all = don't need to make a new one each time - easy to use + adjustable

Question 34

Genome editing by Cas9-CRISPR

Answer 34

1. Cas9 induces site-specific dsb 2. Cell repairs break via 1/2 pathways: NHEJ or HDR

Question 35

Prime editing

Answer 35

Cas9 nickase fused to reverse transcriptase -> makes nick NOT break gRNA fused to RTase = pegRNA = prime editing gRNA - fills in gap with sequence wanted

Question 36

Problem with ds breaks

Answer 36

Can join up with other ds breaks

Question 37

Problem with CRISPR

Answer 37

Not v efficient Depends on cell cycle