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Understanding genetic tools in haematology research - Slide 1

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    Understanding genetic tools in haematology research - Slide 1 Understanding genetic tools in haematology research - Slide 1 Presentation Transcript

    • 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
      • 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?
      • 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 .
      Genetic tools for use in human cells; How?
      • 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