Transcript Mice undergo efficient homologous recombination

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.
 c.f. pharmacological inhibition, isolated
expression systems, etc.
- Widely regarded as the current best practice for proof-ofconcept studies.
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.
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?
- 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).
How to make a knockout mouse
How to make a knockout mouse
- Make your construct & transfect into mouse ES cells:
Select for homologous recombination
How to make a knockout mouse
- 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.
Knockin mice
Uses the same process as making a knockout mouse (nonfunctional 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.
Conditional knockouts
- 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 – 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. ***
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
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).
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 bgalactosidase]. (Disrupts endogenous gene expression - also acts as a
handy reporter.)
Accessible methods for generating knockouts
Genetic tools for use in human cells
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.
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.
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 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.