Lecture 14. Mammalian Cell Engineering 1 Flashcards
What are the main methods of transfection of mammalian cells?
Calcium phosphate
Lipofection
Electroportation
Viral
What is the advantage of calcium phosphate in transfection of mammalian cells?
Cheap, simple, often high efficiency
What is the disadvantage of calcium phosphate in transfection of mammalian cells?
Varying efficiencies in different cell types
What is the advantage of lipofection in transfection of mammalian cells?
Simple, immediate efficiency
What is the disadvantage of lipofection in transfection of mammalian cells?
Varying efficiencies in different cell types
What is the advantage of electroporation in transfection of mammalian cells?
Immediate efficiency
What is the disadvantage of electroporation in transfection of mammalian cells?
Need equipment, stress for cells
What is the advantage of viral in transfection of mammalian cells?
High efficiency (integrate own genome into targeted cell)
What is the disadvantage of viral in transfection of mammalian cells?
Complicated, potentially unsafe
How can you help to achieve stable integration of a plasmid DNA after transfection?
Use a transposase system
How much of the human genome is comprised of transposons?
Roughly 50%
What are the two commercially available types of transposons?
Piggyback and Sleeping Beauty
How does Piggyback transposons work?
Piggyback transposons recognise short sequences flanking the gene of interest (ITR) and if gene of interest is flanked with this, then transposon can incorporate foreign DNA into host DNA
How are the majority of plasmids that get inserted into cells made?
Gibson assembly
How does Gibson assembly work?
Add 3 different enzymes (T5 exonuclease, Phusion polymerase and Taq ligase) and combine them with the DNA fragments you want to join together and that share an overlapping region where you can just do PCR
What is important to remember about Gibson assembly?
That the pairs of fragments share the same overhang on one side
What are examples of the most used activating factors for typical transcription?
VP16 (Virus protein 16), VP64; from Herpes Simplex virus
p65 (NF kB subunit)
E2F4 (E2F family of TFs
What is an example of one of the the most used repressing factors for typical transcription?
KRAB (Krüppel associated box); zinc-finger DBD
How was genome editing before Cas9 carried out?
Recombinase systems
What are the two most important recombinase systems?
Cre/lox
Flp/FRT
What is the recombination site for Cre/lox?
loxP site (reverse compliment)
What happens in the Cre/lox system when two loxP sites face the same direction?
The recombinant excises the DNA flanked by loxP sites and religates the remaining strands of DNA (middle part cut out and removed)
What happens in the Cre/lox system when two loxP sites face each other?
The DNA flanked by loxP is removed, flipped around and reinserted
What happens in the Cre/lox system when two strands of DNA carry one loxP site?
Can produce translocation between the strands, switching elements
What can you do with recombinases examples?
LoxP-flanked, inverted GFP + CD4 promoter driven Cre recombinase in mouse genome ⇒ green labeling of T helper cells (knock-in mouse)
Companies offer common cell lines with loxP/FRT sites at specific location in genome ⇒ easy insertion of single copy of gene
What does a deletion in the CCR5 gene at position 32 cause?
Immunity to HIV
What are the two derivatives of Cas9?
Prime-editing
Base-editing
What happens in prime-editing?
Cas9 is fused to a reverse transcriptase and the guide RNA that is inside the Cas9 is fused to another RNA and serves as the template for reverse transcriptase.
Makes it possible to insert longer strands of new DNA into a certain position in the genome because the reverse transcriptase was reversely transcribed and inserted into the specific location in the genome
What happens in base-editing?
Directly edits the bases of the DNA to change them into different ones.
Transfect editor as mRNA* or protein along with gRNA (RNP) into haematopoietic stem- and progenitor cells (HSPCs)
Tested as a potential cure to sickle cell anaemia
How is base editing superior to Cas9?
It does not inflict damage to the DNA strands
Summary of T cell biology
Derive from thymus (name giving)
Detect antigenic peptides presented by MHC on APCs through TCR
Mostly characterised by surface expression of CD4 or CD8 (MHC I detects CD8, MCH II detects CD4)
Major role in ‘orchestrating’ immune responses
No T cells ⇒ severe combined immunodeficiency (SCID)
What 3 signals activate naive T cells?
Signal 1 - a specific interaction between the TCR and MHC + antigenic peptide
Signal 2 - a co-stimulatory interaction which provides a survival and proliferation signal
Signal 3 - a differentiation signal provided by a secreted cytokine
What are the main strategies of using T cell engineering as therapeutic tool?
Make T cells recognise otherwise undetected antigens (e.g. on tumour cells)
Modulate immune suppression (e.g. tumour microenvironment)
Engineer migration control
What is the CAR T-cell approach
Chimeric Antigen Receptor
Engineer a new type of T cell receptor by combining multiple intracellular signaling domains with single-chain variable fragment (scFv); MHC-independent, high binding strength/specificity
How are scFvs (single-chain variable fragments) generated?
- Make monoclonal antibody
- Sequence Ig locus (You can figure out the exact sequence of what antibody is being made)
- Construct fusion of variable chain and human cytoplasmic domain
How can T cells be engineered?
- Isolate T cells from patient (Apheresis, FACS, MACS)
- Transduce/fect with constructs (gammaretrovirus, lentivirus, electroporation)
- Expand T cells in cell culture by unspecific TCR stimulation & cytokines
- Re-inject into patient
What is apheresis?
Creates gradient of cells and extract the ones you are interested in
What is FACS?
Fluorescent Activated Cell Sorting
Single stream of cells passed through a laser, detector reads out whether there us fluorescence coming from the cell
Cells also get an electric charge and are passed between two electrically charged plates, depending on what charge the cells can be deflected as they pass through the FACS machine, sorts cells by fluorescence
What is MACS?
Magnet Activated Cell Sorting
MACS labels antibodies with iron beads, finds antibodies bound to cells. Purple thing is a magnet. Cells bound to the antibodies/beads are kept in the magnetic fields of the magnet, allowing for you to filter out the cells you don’t want
When is MACS used?
Used to purify CD4 and CD8 cells after doing the immunofluoresence step
