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Understanding genetic tools in haematology research

Understanding genetic tools in haematology research. 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.

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Understanding genetic tools in haematology research

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  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? 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. Obtain human HSCs ↓ Culture into MKs ↓ Silence gene/s ↓ Analysis of function

  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.

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