Daniel Hebenstreit Flashcards
(25 cards)
What are the challenges of mammalian synthetic biology?
- Mammalian cells are complex, with different morphologies and number of nuclei and combinatorial regulation
- Long distance regulation.
- Layers of regulation
- Noise
- Immune system attacks new components you want to integrate
- Isolating cells by cell type is difficult as they die after a few days
What causes noise and why is it a challenge to mammalian synthetic biology?
Burstiness of transcription (lots of RNAs made in short period of time then long periods of silence)
This causes very different mRNA/protein numbers from cell to cell.
It makes it difficult to design circuits, especially with multiple layers of regulation.
Cell division also contributes, partitioning cells unequally. Concentration of proteins also changes as cell size changes over life cycle.
Why does layers of regulation in mammalian cells make synthetic biology challenging?
It is not quite understood how these affect gene expression.
For example histone modifications; mono vs triacetylation is not understood
DNA methylation has not been well studied because it is not found in yeast.
A lot of regulation is also done with small RNAs – mechanisms not fully understood
What is Combinatorial regulation?
When a gene is regulated by multiple transcription factors combined
In a graph which plots number of transcription factors vs number of genes they regulate, different species would have different slopes.
Unicellular ones have a lot steeper curves which implies that COMPLEX organisms have more combinatorial uses of transcription factors.
Give an example of Cas-9 being used.
Announced November 2018 - He Jiankui (chinese)
Cas9 mediated CCR5 deletion was done in human embryos with HIV+ father.
It makes humans less susceptible to HIV infection.
Twin girls called Lulu and Nana
Ethical issues because off target effects are likely, and there are other genes involved so it may not work
What are the two recombinase systems?
Cre/lox recombinase system - Cre recognises Lox sequences
Flp/FRT recombinase system - Flp recognises FRT sequences
Give an example of recombinases being used to study genes required for early development.
In two strains of mice, one expresses recombinase in certain tissues with a specific tissue promoter and the other has a gene flanked with lox sites.
Cross the strains and the gene is knocked out in certain tissues.
What are popular activating domains used in signalling combinatorics?
- VP16 (Virus protein 16); from Herpes Simplex virus
- p65 (NFkB subunit)
- E2F4 (E2F family of TFs)
What is a popular repressor used in signalling combinatorics?
• KRAB (Krüppel associated box); zinc-finger DBD
Give an example of recombinases being used to study genes required for early development.
Two strains of mice; one has loxP-flanked, inverted GFP.
The other has a Cre recombinase fused to a CD4 promoter(CD4 is expressed in T helper cells only).
Crossing the strains means that Cre recombinase is only expressed in T helper cells so GFP becomes active in these cells only.
What do you need to remember/be careful of with recombinases?
To turn the system off once the desired outcome is achieved to prevent further recombination
What is signalling combinatorics?
It combines all the techniques, gibson assembly, recombinases and crispr to engineer novel signalling pathways. and control transcription of existing pathways.
Give an example of a signalling combinatorics in use
Degrones, which facilitate ubiquitination, can be made to be inducible by small molecules
Why are mammalian cells difficult to work with?
hard to transfect
How can you transfect mammalian cells?
- Calcium phosphate = easy and efficient but doesn’t work in some cell types.
- Lipfection = simple but not efficient in all cell types
- Electroproation = needs equipment and is stressful for cells
- Viral = highly efficient but complicated and potentially dangerous as viruses may have off target effects.
What are CA positive T cells?
Ones with cytotoxic activity
What disease shows the function of T cells?
SCID - severe combined immunodeficiency
What are CAR T cells?
T cells with Chimeric Antigen Receptors, which have been engineered to have new antigen specificity
What are CAR T cells?
T cells with Chimeric Antigen Receptors
What are the main strategies of T cell engineering as a therapeutic tool?
- make T cells recognize otherwise undetected antigens (e.g. on tumor cells)
- modulate immune suppression (e.g. tumour microenvironment)
- Engineer migration control
How do you make CARs?
Take an scFV fragment from an antibody that detects the desired antigen and engineer a fusion with a signalling domain. This can be used to make T cells attack tumours; it detects antigens in an MHC independent fashion
How do you make CARs?
Take an scFV fragment from an antibody that detects the desired antigen and engineer a fusion with a signalling domain
How do you generate scFVs?
- Make monoclonal antibody of the desired antigen
- Sequence a lmmunoglobulin locus (
- Construct fusion of variable chain and human cytoplasmic domain – clone something to make it the top part of a receptor?
How do you make monoclonal antibodies?
Antiserum is made by challenging a mouse with a pathogen and taking their blood.
It will contain antibodies which bind the antigen at varying strengths from not at all to very strongly.
Monoclonal antibodies come from a single clone of B cells.
Immunise animals with the antigen you want antibodies for. Take the animal’s spleen cells and dilute down. Fuse with myeloma cells to make them immortal. Test and select cells which make the best antibodies.
If they all come from a single cell then they are clonal so antibodies that come from them are monoclonal.
Take the protein sequence of the antibody to use as the CAR
