1. Understanding genetic tools in haematology research

2. Why use genetics?
- To investigate the function of a protein/s of interest.
Examine (patho)physiological processes in the absence of this protein.
Provides a test of unparalleled cleanliness and specificity.
 c.f. pharmacological inhibition, isolated expression systems, etc.
- Widely regarded as the current best practice for proof-of-concept studies.

3. The rise and rise of the mouse as a model
Mice undergo efficient homologous recombination
Allows replacement of an allele with an engineered construct.
Used for creating knockout and knockin mice.

4. Why make a knockout mouse?
- To investigate the function of a protein/s of interest.
Lack of well-characterised pharmacological tools.
To allow thorough in vivo analysis of the function of YFP in both spontaneous and induced phenotypes.
If you have a strong hypothesis!

5. Why make a knockout mouse?
- To investigate the function of a protein/s of interest.
Lack of well-characterised pharmacological tools.
To allow thorough in vivo analysis of the function of YFP in both spontaneous and induced phenotypes.
If you have a strong hypothesis!
Examples in haematology:
Platelet receptors (e.g. thrombin receptors), coagulation factors (e.g. FII, FXII), coagulation modulators (protein Z, TM).

6. How to make a knockout mouse

7. How to make a knockout mouse
- Make your construct & transfect into mouse ES cells:
Select for homologous recombination

8. How to make a knockout mouse
- Inject mutant ES cells into blastocysts and transfer these to psuedo-pregnant female mice.

9. How to make a knockout mouse
- Screen by coat colour and then by transmissibility.

10. Knockin mice
Uses the same process as making a knockout mouse (non-functional allele) but generally replaces or adds a gene.
Can therefore be used for gain-of-function studies.
Examples include:
- Humanising a protein in a mouse;
Introducing a point mutation (e.g. to model a human condition or to determine functions of specific protein motifs);
Stable introduction of a marker or experimental tool into the genome.

11. Conditional knockouts
- Aims to exert a level of spatial and temporal control over the removal of genes.
- Most commonly used to
Overcome a gross phenotype in global gene deficiency
(e.g. embryonic lethality, perinatal haemorrhage) or
ii) Dissect cell-specific contributions to multicellular disease states.
Involves an enzyme-based removal of genomic DNA in cell type/s of interest.

12. Conditional knockouts – the lingo
Cre/loxP = the most commonly used system for conditional gene excision.
(FLP/FRT is another.)
Cre = a site-specific DNA recombinase from bacteriophage.
loxP = recognition sites for Cre recombinase.
*** The specificity of gene excision is determined by the promoter used to control expression of Cre. ***

13. Conditional knockouts:
Use in haematology research
Most commonly used Cre mouse lines in haematology are:
- Tie2-Cre (v. early endothelial and therefore also haematopoietic).
- Vav-Cre (haematopoietic-specific, low/no endothelial excision).
- PF4-Cre (one-and-only platelet-specific line).
- Mx1-Cre
- interferon-responsive promoter.
- allows ‘external’ temporal control over Cre expression.
- pan-haematopoietic.

14. Conditional knockouts:
Use in haematology research
Most commonly used Cre mouse lines in haematology are:
- Tie2-Cre (v. early endothelial and therefore also haematopoietic).
- Vav-Cre (haematopoietic-specific, low/no endothelial excision).
- PF4-Cre (one-and-only platelet-specific line).
- Mx1-Cre
- interferon-responsive promoter.
- allows ‘external’ temporal control over Cre expression.
- pan-haematopoietic.
Examples in haematology:
Transcription factors (e.g. SCL), ubiquitous signalling proteins (e.g. G proteins), coagulation factors (TF).

15. Accessible methods for generating knockouts
- Average knockout costs ~$40K and takes ~1.5 yr to generate.
International knockout mouse project aims to delete all ~ 30,000 mouse genes in ES cells.
Gene trap-mediated insertion [of promoterless gene for b-
galactosidase]. (Disrupts endogenous gene expression - also acts as a handy reporter.)

16. Accessible methods for generating knockouts

17. Genetic tools for use in human cells

18. Genetic tools for use in human cells:
Why?
Genetics is a powerful tool for investigating the functions of proteins of interest and has been widely used in haematology-related research.
For this field, it is currently limited to fish and mice (and naturally occurring human conditions).
One challenge for the field is how best to advance from the era of mouse genetics.

19. Genetic tools for use in human cells;
How?
RNA-mediated interference (RNAi):
Naturally occurring mechanism for regulating gene expression.
dsRNA inhibits the expression of genes with complementary nucleotide sequences.
Occurs in most eukaryotes, including humans.
Synthetic dsRNA introduced into cells in culture can induce suppression of specific genes of interest.
New methods allow stable and selectable expression of “dsRNA” in cells of interest.

20. Genetic tools for use in human cells;
How?
One goal is to establish a system whereby selected genes can be specifically down-regulated in human MKs/platelets for the purpose of examining protein function in vitro.

21. Genetic tools for use in human cells;
How?
Obtain human HSCs ↓
Culture into MKs
Silence gene/s
Analysis of function

22. Genetic tools for use in human cells;
How?
Obtain human HSCs ↓
Culture into MKs
Silence gene/s
Analysis of function
Antibody-based (CD34+) isolation from peripheral blood leukocytes taken from mobilised patients undergoing harvest for transplantation.
Culture in presence of Tpo (+/- Epo, IL-3, SCF) for maturation into >90% MK.
Transfect with lentivirus producing shRNA against you target of interest.
For platelets: Aggregation, secretion, IIbIIIa activation.
For MKs: Ca2+ and other signalling events, IIbIIIa activation.

23. Genetic tools for use in haematology research
Wide application.
Many past successes.
Not as technically prohibitive as it used to be.
Translation of genetic techniques to human systems happening now.
Significant scope for clinical research application.