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Understanding genetic tools

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How to make a knockout mouse ... used Cre mouse lines in haematology ... leukocytes taken from mobilised patients undergoing harvest for transplantation. ... – PowerPoint PPT presentation

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Title: Understanding genetic tools


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 gt90 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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