Gene edit/animal models

Question 1

How could you introduce DNA into the bacteria?

Answer 1

Micro injection
Viral transfection
Heat shock
Electroporation

Question 2

What are the limitations of using E. coli ?

Answer 2

Limited post translational modification eg. No glycosylation
Improper folding
Degradation
Lack of ability to perform intron-exon splicing
Codon bias- difficulties in host tRNA translating the mRNA
Foreign genes may contain bacterial termination signals

Question 3

What are the pros and cons of using yeast in cloning?

Answer 3

Is eukaryotic so will be more homologous than E. coli- post translational modification and protein degradation
High yields, fast, cheap and easy. Genome fully sequenced and understood.
Many human homologues to yeast mutations.
However, it is unicellular so it is not a specialised secretory cell

Question 4

What are the pros and cons of using mammalian cells?

Answer 4

Difficult, slow, costly and cells require a surface to grow on rather than a suspension
If gene is over expressed it can have deleterious effect on the cell so it will be down regulated
But the protein can be accurately post translationally modified and spliced etc.
Far similar genome to humans + associated mutations.

Question 5

Describe vectors

Answer 5

A vehicle used to transfer genetic material to a target cell
It needs to be capable of independent replication within a host and of carrying a fragment of foreign DNA
Eg. Plasmids- 10kb, bacteriophages lambda phage-23kb, cosmids 30-44kb, YACS 0.2-2Mb, BACS 300kb

Question 6

What is gene editing?

Answer 6

Enables manipulation of virtually any gene in a diverse range cell types and organisms
Based in the use of engineered nucleases

Question 7

Outline the methods of genome editing

Answer 7

Zinc-finger nucleases (ZFNs)- zinc fingers recognise a codon- attached to Fok1 which cleaves in pairs- two different series of zinc-fingers are required on either side of the cleavage site
Transcription Activator-Like Effector Nucleases (TALENS)- also uses Fok1 but uses repeat variable diresidues (RVD) to recognise individual nucleotides- one either side of the cleavage site
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- Cas9 is the nuclease, requires tracrRNA and crRNA- cleaves and guides sequence on the crRNA into genome.

Question 8

Limitations of zinc fingers

Answer 8

• poor targeting density
• methylation sensitive
• not all newly synthesised ZFNs are able to cleave chromosomal DNA
• low success rate
• has a high amount of off target effects
• costly and difficult to produce and deliver

Question 9

Give an overview of using TALENS for gene editing

Answer 9

• Can be used to recognise small sequences
• High success rate
• Low off target effects
• Less costly to produce
  Limitations
• large constructs
• Highly homologous- self-recombination
• 5’ thymine and methylation sensitive

Question 10

Give an overview of using CRISPR-Cas9 for gene editing

Answer 10

• Has a high success rate
• Capable of multiplexing
• Low cytotoxicity
• Affordable and easy to modify/deliver
  Limitations
• restriction site limitations- end with an NGG or NAG
• Has variable off target effects
• Big construct

Question 11

How could you deliver reprogrammable nucleases?

Answer 11

Via plasmid DNA 
In vitro transcribed mRNA
Non-integrating viral vectors 
Purified protein
➡️
Electroporation
Liposome transfection
Micro injection

Question 12

What are the applications of genome editing?

Answer 12

Pre-clinical
-Targeting non-coding regions
-Creation of genetic variation and study their effects
-Generation of knock-out and knock-in models by direct injection into embryos
-Create an isogenic cell line to model human disease
Biotechnology
-GM crops and livestock
-Production of therapeutic proteins in cultured cell lines
Therapeutics
-Treatment of HIV in humans
-Treatment of haemophilia B in mice
-Gene correction and addition in patient-derived pluripotent stem cells for patients with genetic diseases

Question 13

What cell activity does genome editing rely on?

Answer 13

Double strand break repair
NHEJ repair can lead to deletions or inserting that can knock out a gene
Insertion of donor DNA, single nucleotides or tags

Question 14

Describe how HIV can be treated with genome editing

Answer 14

People homozygous for the CCR5delta32 mutation of the CD4 T cell receptor are resistant to HIV
ZFN/NHEJ knock out of CCR5 in humanised mouse model of HIV showed reduced viral load and improved CD4 T cell counts

Question 15

What makes a good model organism?

Answer 15

Developmental anatomy
Size and complexity of housing required
Generation time
Ease of study- genome sequence, size, mammal, non-mammal embryo models
Transparency
Organismal complexity or simplicity
Ethical and legal considerations- reduction, refinement, replacement

Question 16

Define the three Rs of animal research

Answer 16

Replacement- methods to avoid or replace the use of animals
Eg. Human volunteers, tissues and cells
Mathematical models
Established cell lines
Immature forms of vertebrates or invertebrates
Reduction- methods which minimise the number of animals used per experiment
Eg. Improved experimental design and statistical analysis, sharing data and resources
Use if imaging technologies to enable longitudinal studies in the same animal
Refinement- methods to minimise suffering and improve animal welfare
Eg. Appropriate anaesthetics and analgesics, avoiding stress
Using appropriate housing that allows the expression if species specific behaviours

Question 17

Why are Drosophila melanogaster a good model?

Answer 17

65% of known human disease loci have clear homologues in Drosophila
More than 100 yrs of genetic research has generated powerful tools- first genome to be sequenced and annotated
Excellent model for discovering gene function and cellular pathways
Complex nervous system and behaviours

Question 18

How would you analyse gene function in Drosophila?

Answer 18

1. Make a mutant (loss of function)- imprecise excision of P-elements, homologous recombination
2. Overexpress the gene (gain of function, eg. The Gal4-UAS system
3. Knockdown gene excited expression (RNAi) eg. UAS dsRNA X Gal4 driver leads to tissue specific knockdown

Question 19

What are the most common vertebrate models of human development?

Answer 19

Clawed frog- Xenopus laevis/tropicalis 
Chick- Gallus gallus 
Mouse- Mus musculus 
Rat- Rattus norvegicus 
Zebrafish- Danio rerio
Medaka- Oryzias latipes

Question 20

Describe the usefulness of using zebrafish for human models

Answer 20

Vertebrate
Transparent
Large numbers
Ease of micro manipulation, grafting, filming, phenotype screening
Forward genetics
Fast 'gene knock down' 
Knock out with ZFN and Crispr Cas9
Genomic resources

Question 21

How would you generate animal models if disease?

Answer 21

Spontaneous mutants- hard to find
Eg. Haemophiliac dog, loss of function IX
Watanabe heritable hyperlipaemic rabbit- familial hypercholesteraemia
Induced mutagenesis- forward genetics
-chemical treatment or irradiation of sperm
But may soon us random an screening laborious
Transgenic models- reverse genetics

Question 22

Describe the mouse as an experimental system

Answer 22

Genome- 99% of its ~25000 genes have a human counterpart
Life cycle- 4 day oestrus, 20 day gestation, 4-8 pups per litter, 2-8 litters per female, 7 weeks to sexual maturity, 2-3 yr lifespan
Reverse genetics- knockouts transgenics, conditional expression, inducible expression, retroviral vectors, siRNA
Strains- inbred, out red, recombinant inbred, consomic, fluorescent
Assisted reproduction- cryopreservation, embryo rederivation, in vitro fertilisation, intracytoplasmic sperm injections, cloning
Tools- genome sequencing, embryonic stem cells, expression arrays, gene trap libraries, insertional vector libraries, BAC libraries

Question 23

How can you create a transgenic mouse?

Answer 23

1. Build transgene construct
2. Introduce into cell (zygote or ESC)
3. Integrate DNA into genome
4. Select transgene carriers- embryo➡️mosaic or ESC➡️chimaera
5. Implant into foster mother
6. Breed chimaeric offspring to select for heterozygotes
7. Make homozygous by sister crossing

Question 24

Describe the transformation of cells/zygotes

Answer 24

Transduction- virally mediated gene transfer- AAV naturally integrates into the genome
Transfection- chemical or electric mediated mediated gene transfer- CaPO4, lipid complexes, Electroporation
Direct transfer- micro injection, particle bombardment

Question 25

Describe the implantation of the transgenic zygote/embryo

Answer 25

Needle is used to introduce zygotes into the uterus of a pseudopregnant recipient female
(Copulated with vasectomised male)

Question 26

What is the advantage of using homologous recombination for transgene integration?

Answer 26

Can target site
Replace genes
Knock out

Question 27

Describe the positive and negative selection of targeted ES cells

Answer 27

Some cells will be non-recombinant, some recombinant with random inserts and some recombinant a with gene target insertion

1. Selection for antibiotic resistance marker gene eg. Neor- neomycin removes non recombinants
2. Selection for gene that should have been removed by targeted homologous recombination eg. TK- ganciclovir removes the random insert recombinants

Question 28

What is a conditional knock out?

Answer 28

The target gene can be eliminated in a single organ
Cre/IoxP mediated gene deletion system
Cre recombinase recognises IoxP flanked sequences
It is only expressed if there is a tissue specific promoter

Question 29

Cre/LoxP

Answer 29

Cre Recombinase is a site specific recombinase that recognises the loxP sequence. Introduce two loxP sites around a gene of interest.
If Cre recombinase is introduced it will cut and remove the gene of interest. Allows for driving of mutation when and where the Cre recombinase is available.
Cre recombinase can be regulated by a tissue specific promoter, such as a skin promoter. A mouse with the Cre-recombinase and tissue specific promoter is crossed with a mouse that has the loxP sequences around the target gene.
Cre will be expressed in a tissue specific manner and so knockout the gene in a tissue specific manner. The gene can also be temporally regulated by hormones and their nuclear receptor so the gene can be knocked out at a specific time during development.
The Cre will only enter the nucleus and knock the gene out by cutting loxP when the hormone is present, as this is the only time it is able to enter the nucleus.