Limitations of germline tansgenesis and newer methods Flashcards
What are the limitations of germline transgenesis?
… how have these limitations been overcome
Limitations that can occur in the creation of transgenic animals is that they are ‘reactionist’.
Therefore, changes in gene function may be due to:
- the transgene introduced
- how the particular strain of animal responds to the transgene introduced
- the environment
Further limitations consist of not knowing what to particularly search for and the difficulty in finding cell lines ( neurons for example don’t divide and so especially difficult to observe neural cell line.
Methods have been designed to overcome these limitations, through:
1) Inducible expression - can result in inducible knockout
2) Target gene expression and repressible transgenesis
3) cell-specific gene expression/knock outs
What is loxP-Cre?
Structure and mechanisms
Using bacteriophage recombination to induce cell-specific knock outs
2 components:
LoxP
Cre: mediates LoxP recombination
LoxP structure:
- two 13bp sequences either side of a 8bp sequence: creates asymmetry which gives directionality
- palandromic sequence needed for:
1) integration into host genome
2) replication
Mechanisms for replication
1) dimerisation of 2 linear phage DNA (linear phage DNA circularises via LoxP sites (at the redundant terminals)
Note: LoxP sites pointing at the same direction
3) Cre mediated recombination of circular dimer creates two separate circular monomers - each containing 1 loxP site
What are the uses of LoxP-Cre?
1) Cell specific knockouts
2) targeted gene activation
3) chromosomal translocation
4) precise mutations
Through: inversions, deletions and translocation
How to detect target gene activation using LoxP-Cre?
Monitor gene expression in vivo via microPET
1) Create mouse with Cre fused to NLS (nuclear localisation sequence, to ensure entry into nucleus): under the control of a cell-specific promoter e.g 2-MHC for myocytes
3) Insert adenoviral vector that has stop sequences flanked by LoxP and HSV1-TK
4) if Cre is being expressed, it will interact with LoxP to mediate recombination.
- This will remove transcriptional stop sequence allowing for expression of HSB1-TK
5) Use microPET to detect TK
How to do cell-specific knock out using LoxP-Cre?
1) Create targeting vector which consists of a targeting cassette: that has neomycin selectable marker (neor) and thymidine kinase (TK) flanked by LoxP
2) Transfect ES cells and select for homologous recombination by neomycin selection in the presence of G418
3) Expression of Cre: choose for type 2 deletion
4) Transfer into blastocyst
5) mate loxP animal with a mouse that has Cre ‘under the control’ of a cell-specific promoter
6) Double transgenic animal created (has both LoxP and Cre): which has cell-specific targeted gene knockout
What is TET on/off used for?
Steps…
TET on/off mechanism is to allow for inducible expression in living adult animals
The TET system of the bacteria has been manipulated….
TET-off:
Expression is off in the presence of doxycycline
1) The TET repressor has been mutated by being fused to VP16 (herpes virus)
2) Therefore, when the mutated repressor is bound to the TET response element (TRE), you get expression
3) When doxycycline is added, it binds to mutated TET, resulting it to fall off from TRE and expression to stop.
Tet -on:
Is the reverse of this system, which has been mutated to ensure that expression starts only in the presence of doxycycline
Doxycycline can be fed to adult mice via water as it passes through the blood brain barrier
How to create inducible cell- specific knockouts?
1) Triple transgenic animals:
By mating animals that have been modified via: TET-on, Cre and LoxP
2) Tamoxifen induced knockout:
- Cre is fused to glucocorticoid receptor which is sequestered in the cytosol
- Tamoxifen binds to ER, it looses its chaperones and translocates to the nucleus
Alternatives to creating knockouts?
1) Cell ablation
- Destroying cells, letting the animal recover and seeing behavioural changes
- used to be through creating lesions
- diphtheria toxin: ‘Amp chain blocks EF2, stopping translation and causing cell death
2) recombinant Antibodies
- blocks activity of proteins via binding
3) expression of dominant negative mutants
- proteins work as dimers… so create a mutant from of protein targeted wild type protein dimerises with. Binding will block activity
4) expression of antisense RNA
- complementary to target RNA to hybridisation stops translation, splicing or mediates degradation via RNase H
What is gene editing?
How is it achieved…
Gene editing is the ‘insertion, deletion or replacing of DNA’ in living organisms using targeted nucleases
- They create specific double stranded break
- dsDNA break is repaired by endogenous repair mechanisms through either:
1) non homologous end joining
- insertion or deletion of base pairs (thus, gene disruption) which results in random mutations
2) homology-directed repair
Preferred method as it creates precise modifications through using a donor DNA template
Zinc nuclear fingers … what is this method about?
Structure, mechanisms, advantages and limitations
Zinc finger nucleases are:
- composed of zinc finger arrays and non-specific nuclease domain derived from Fokl
- each zinc finger can identity 3-4 bp. More can be strung together for the purpose of identifying longer sequences ensuring specificity
- limitations consist of the fact that it’s labour intensive and dependent on interaction of neighbouring fingers
CRISPR/Cas9
Structure and mechanisms?
Clustered regularly interspersed short palindromic sequences
3 steps for how it functions,
1) Acquisition: Viral DNA fragmented and inserted into CRISPR array
2) Expression of crRNA which is palindromic to viral DNA
3) Interference: in future viral event… crRNA will recognise viral DNA and Cas locus will cleave it
Structure consists of:
crRNA: 20 nucleotide guide sequence complementary to target gene
tracrRNA: transactivating crRNA -complementary to CRISPR repeats to facilitate binding to crRNA- forming a gRNA
- tracRNA is needed for crRNA generation and cleavage
gRNA complex fuses to Cas9: forming the Cas9:gRNA complex necessary for cleaving targets
Methods for delivering CRISPR/Cas9?
DNA ( could cause off target mutations), RNA, Proteins
Vectors… non-viral and viral
Neuronal… through viral vector or in utero electroporation
Limitation with neuronal delivery is that neurons don’t divide: thus only do NHEJ Homology directed repair could be promoted through the introduction of a ssDNA donor or therapeutic approaches
It’s possible to make Cas9 cell-specific through the use of cell specific promoters to drive expression
How does CRISPR/Cas9 cleave targets?
1) cleaves 3bp 5’ to PAM sequences
PAM: protospacer adjacent motif
2) is a limitation because you have to choose a genome that has PAM sequence and it changes dependent on the Cas9 protein species
Advantages and disadvantages of CRISPR/Cas9?
Advantages:
- works on many species
- can generate complex mutations
- is highly specific
- it’s an easy and fast method
- is better than miRNAs because it targets all types of DNA
Disadvantages:
- Requires presence of a PAM sequence
- NHEJ is more common in most cells
- potential off targets which can be reduced by…
… limiting levels of CAS9, too much results in off target mutations
- inducible Cas9 expression
- direct delivery of Cas9:gRNA which results in transient Cas9
What is somatic transgenesis?
Aspects to take into consideration…
Somatic transgenesis involves introducing genes into cells other than egg or sperm (in living animals)
for the treatment of illnesses caused by defective genes
Aspects to consider:
- specific cell populations have to be targeted (as it may be toxic to cells that don’t need it)
- long term expression (to ensure patients don’t have to keep going though procedures)
- agent used needs to be capable of inserting genetic material into target cell
- a simple method of delivery (not too invasive)
- able to regulate transgene (many genes are controlled by the circadian rhythm, transgene needs to be able to be turned on or off)
