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Understanding genetic tools
in haematology research
- 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.
Why use genetics?
Mice undergo efficient homologous recombination
The rise and rise of the mouse as a model
- Allows replacement of an allele with an engineered construct.
- Used for creating knockout and knockin mice.
- 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!
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!
Why make a knockout mouse?
Examples in haematology:
Platelet receptors (e.g. thrombin receptors), coagulation factors (e.g. FII, FXII), coagulation modulators (protein Z, TM).
How to make a knockout mouse
- Make your construct & transfect into mouse ES cells:
How to make a knockout mouse
Select for homologous recombination
- Inject mutant ES cells into blastocysts and transfer these to psuedo-pregnant female mice.
How to make a knockout mouse
- Screen by coat colour and then by transmissibility.
How to make a knockout mouse
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.
Knockin mice
- Aims to exert a level of spatial and temporal control over the removal of genes.
- Most commonly used to
i) 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.
Conditional knockouts
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. ***
Conditional knockouts – the lingo
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.
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.
Conditional knockouts:Use in haematology research
Examples in haematology:
Transcription factors (e.g. SCL), ubiquitous signalling proteins (e.g. G proteins), coagulation factors (TF).
- 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 - galactosidase]. (Disrupts endogenous gene expression - also acts as a
handy reporter.)
Accessible methods for generating knockouts
Accessible methods for generating knockouts
Genetic tools for use in human cells
• 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.
Genetic tools for use in human cells:Why?
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.
Genetic tools for use in human cells;How?
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.
Obtain human HSCs
↓Culture into MKs
↓Silence gene/s
↓Analysis of function
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.
Genetic tools for use in human cells;How?
• 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.
Genetic tools for use in haematology research
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