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Understanding genetic tools in haematology research - Slide 1

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  • 1. Understanding genetic tools in haematology research
  • 2. - 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?
  • 3. 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.
  • 4. - 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?
  • 5. - 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).
  • 6. How to make a knockout mouse
  • 7. - Make your construct & transfect into mouse ES cells: How to make a knockout mouse Select for homologous recombination
  • 8. - Inject mutant ES cells into blastocysts and transfer these to psuedo-pregnant female mice. How to make a knockout mouse
  • 9. - Screen by coat colour and then by transmissibility. How to make a knockout mouse
  • 10. 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
  • 11. - 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
  • 12. 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
  • 13. 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
  • 14. 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).
  • 15. - 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 β- galactosidase]. (Disrupts endogenous gene expression - also acts as a handy reporter.) Accessible methods for generating knockouts
  • 16. Accessible methods for generating knockouts
  • 17. Genetic tools for use in human cells
  • 18. • 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?
  • 19. 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?
  • 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. Obtain human HSCs ↓ Culture into MKs ↓ Silence gene/s ↓ Analysis of function Genetic tools for use in human cells; How?
  • 22. 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?
  • 23. • 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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