understanding genetic tools in haematology research - slide 1

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.


- 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?


- 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:


Select for homologous recombination

- Inject mutant ES cells into blastocysts and transfer these to psuedo-pregnant female mice.


- Screen by coat colour and then by transmissibility.


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

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

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.

Obtain human HSCs

↓Culture into MKs

↓Silence gene/s

↓Analysis of function


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.


• 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

understanding genetic tools in haematology research - slide 1

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