Lecture 14. Mammalian Cell Engineering 1

Question 1

What are the main methods of transfection of mammalian cells?

Answer 1

Calcium phosphate
Lipofection
Electroportation
Viral

Question 2

What is the advantage of calcium phosphate in transfection of mammalian cells?

Answer 2

Cheap, simple, often high efficiency

Question 3

What is the disadvantage of calcium phosphate in transfection of mammalian cells?

Answer 3

Varying efficiencies in different cell types

Question 4

What is the advantage of lipofection in transfection of mammalian cells?

Answer 4

Simple, immediate efficiency

Question 5

What is the disadvantage of lipofection in transfection of mammalian cells?

Answer 5

Varying efficiencies in different cell types

Question 6

What is the advantage of electroporation in transfection of mammalian cells?

Answer 6

Immediate efficiency

Question 7

What is the disadvantage of electroporation in transfection of mammalian cells?

Answer 7

Need equipment, stress for cells

Question 8

What is the advantage of viral in transfection of mammalian cells?

Answer 8

High efficiency (integrate own genome into targeted cell)

Question 9

What is the disadvantage of viral in transfection of mammalian cells?

Answer 9

Complicated, potentially unsafe

Question 10

How can you help to achieve stable integration of a plasmid DNA after transfection?

Answer 10

Use a transposase system

Question 11

How much of the human genome is comprised of transposons?

Answer 11

Roughly 50%

Question 12

What are the two commercially available types of transposons?

Answer 12

Piggyback and Sleeping Beauty

Question 13

How does Piggyback transposons work?

Answer 13

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

Question 14

How are the majority of plasmids that get inserted into cells made?

Answer 14

Gibson assembly

Question 15

How does Gibson assembly work?

Answer 15

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

Question 16

What is important to remember about Gibson assembly?

Answer 16

That the pairs of fragments share the same overhang on one side

Question 17

What are examples of the most used activating factors for typical transcription?

Answer 17

VP16 (Virus protein 16), VP64; from Herpes Simplex virus
p65 (NF kB subunit)
E2F4 (E2F family of TFs

Question 18

What is an example of one of the the most used repressing factors for typical transcription?

Answer 18

KRAB (Krüppel associated box); zinc-finger DBD

Question 19

How was genome editing before Cas9 carried out?

Answer 19

Recombinase systems

Question 20

What are the two most important recombinase systems?

Answer 20

Cre/lox
Flp/FRT

Question 21

What is the recombination site for Cre/lox?

Answer 21

loxP site (reverse compliment)

Question 22

What happens in the Cre/lox system when two loxP sites face the same direction?

Answer 22

The recombinant excises the DNA flanked by loxP sites and religates the remaining strands of DNA (middle part cut out and removed)

Question 23

What happens in the Cre/lox system when two loxP sites face each other?

Answer 23

The DNA flanked by loxP is removed, flipped around and reinserted

Question 24

What happens in the Cre/lox system when two strands of DNA carry one loxP site?

Answer 24

Can produce translocation between the strands, switching elements

Question 25

What can you do with recombinases examples?

Answer 25

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

Question 26

What does a deletion in the CCR5 gene at position 32 cause?

Answer 26

Immunity to HIV

Question 27

What are the two derivatives of Cas9?

Answer 27

Prime-editing Base-editing

Question 28

What happens in prime-editing?

Answer 28

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

Question 29

What happens in base-editing?

Answer 29

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

Question 30

How is base editing superior to Cas9?

Answer 30

It does not inflict damage to the DNA strands

Question 31

Summary of T cell biology

Answer 31

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)

Question 32

What 3 signals activate naive T cells?

Answer 32

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

Question 33

What are the main strategies of using T cell engineering as therapeutic tool?

Answer 33

Make T cells recognise otherwise undetected antigens (e.g. on tumour cells) Modulate immune suppression (e.g. tumour microenvironment) Engineer migration control

Question 34

What is the CAR T-cell approach

Answer 34

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

Question 35

How are scFvs (single-chain variable fragments) generated?

Answer 35

1. Make monoclonal antibody 2. Sequence Ig locus (You can figure out the exact sequence of what antibody is being made) 3. Construct fusion of variable chain and human cytoplasmic domain

Question 36

How can T cells be engineered?

Answer 36

1. Isolate T cells from patient (Apheresis, FACS, MACS) 2. Transduce/fect with constructs (gammaretrovirus, lentivirus, electroporation) 3. Expand T cells in cell culture by unspecific TCR stimulation & cytokines 4. Re-inject into patient

Question 37

What is apheresis?

Answer 37

Creates gradient of cells and extract the ones you are interested in

Question 38

What is FACS?

Answer 38

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

Question 39

What is MACS?

Answer 39

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

Question 40

When is MACS used?

Answer 40

Used to purify CD4 and CD8 cells after doing the immunofluoresence step