Lecture 3: Getting DNA into cells Flashcards
How can we transform bacteria with heat shock?
Bacterial transformation is the process of adding DNA into bacterial cells and ensuring that only transformed cells grow.
1) Make cells competent. Add RbCl and calcium chloride. Incubate at 4 degrees Celsius.
2) Add DNA.
3) Heat shock at 42 degrees Celsius for 10 seconds to a minute.
4) DNA is taken up.
5) Incubate in LB for 30 minutes. During this time the antibiotic resistance gene should be expressed.
6) Antibiotic is added. Selection for transformed cells.
What are the pros and cons of heat shock?
Pros • No specialised equipment. • DNA volume doesn’t matter • Can tolerate salt in DNA preparation. • Generally reliable.
Cons
• Takes longer.
• Lower transformation efficiency.
What are the pros and cons of electroporation?
Pros
• Quick
• Low DNA volume
• Higher transformation efficiency.
Cons
• Variable outcomes. Can work very well.
• Expensive equipment and consumables.
• Very salt sensitive.
What are the issues with using bacteria for eukaryotic proteins?
- Codon differences. Leads to premature chain termination. Can be overcome with Rosetta.
- Possible protein folding issues. Overcome with Origami.
- Targeting to membranes or organelles isn’t possible. Extra tags needed for secretion.
- Serine/threonine/tyrosine phosphorylation does not happen but may be essential to protein function (e.g. p53 DNA binding activity).
- No glycosylation.
How do we culture mammalian cells?
We can culture in many different ways:
• Monolayer monoculture.
• Cell factories. For protein production.
• Cell layers (in scaffolds) to form tissue/organs.
• Organoids.
Certain cells are isolated from tissues such as fibroblasts from skin, adipocytes and hepatocytes. They then require specific conditions.
• 37 degrees. 5% carbon dioxide, 2-20% oxygen and a humidified incubator.
• Nutrients and growth factors. We need nutrients like pyruvate, glucose and glutamate. Grow in a buffered medium (e.g. DMEM).
• Primary cells stop dividing when they touch (contact inhibition). Hence we need to refeed, harvest and replate regularly.
• Monitor proliferation with live imaging and cell counting.
• Cryogenically preserve cells in liquid nitrogen for future culture.
How does microinjection work?
Directly transfers DNA using a micropipette with a DNA solution. Another pipette holds the egg in place.
What is sonoporation?
Sonoporation is a technique which is used for getting DNA into whole organisms. Disrupts the membrane and creates microbubbles to enhance delivery of large molecules.
How can we use calcium phosphate transfection?
CaPO4 transfection can be used to cheaply and easily insert DNA into eukaryotes.
• Mix HEPES-buffered saline with calcium chloride and DNA to be transfected.
• CaPO4¬ precipitates and DNA binds to the surface of it.
• Mammalian cells take up liquids from their environment by micropinocytosis.
• Tiny particles are also taken up in pinocytic vesicles. The precipitates are taken up.
• Low efficiency.
What is liposomal transfection?
Liposomes are lipids which can surround DNA.
• Very efficient (around 90%).
• Fusion of lipid with the plasma membrane releases DNA into the cytoplasm.
• Highly toxic, elicits innate immune response (it shouldn’t be there).
• Some cells (e.g. primary cells) are hard to transfect. Polyamines can help (lipofectamine). *
• Not very effective in many cells.
What is a gene gun?
The gene gun physically places DNA into cells with a particle gun.
• Tiny DNA coated metal particles called microprojectiles are suspended in a drop on a macroprojectile.
• An explosion propels the macroprojectile forward until it hits the stopping plate. Microprojectiles keep going forward.
• DNA introduced with the particles is expressed.
• Can be used in transient gene expression assays. Does the gene construct function?
• Can also be used to make permanently transformed cell lines or organisms e.g. C. elegans.
• Only technique which has successfully put gene in plastids and mitochondria.
• Can be used in genetic immunisation by impelling genes for antigens into an animals skin (powderject).
How can we use bacteria for GM plants?
This technique is based on crown gall tumour-causing bacterium Agrobacterium tumefaciens.
• Ti DNA required for infection. Ti plasmid DNA has auxins. **
• Make plasmid with GOI in E. coli.
• Transformed bacterium with the plasmid.
• Infect cultured plant cells with engineered bacteria.
• Grow up the whole plant from individual infected cells.
What are adenoviruses and adeno-associated virus vectors? What are the advantages?
Adenoviruses have lower toxicity than chemical transfection or electroporation.
1) Clone foreign gene into shuttle vector.
2) Recombine with AdEasy in E. coli.
3) Isolate plasmid and linearise.
4) Liposomal transfection of HEK293s.
5) Productive infection gives a virus that can infect other cell types.
They have a number of advantages
• Adenoviruses can have up to 100% transfection efficiency, even in hard to transfect cells.
• Infect both cycling and non-cycling cells.
• Reasonable insert capacity. Low cancer risk as low integration rates.
• AAVs are even better.
However, there is no genomic integration.
What are retroviruses?
Retroviruses are RNA viruses. There are 3 outcomes which can occur when they infect cells.
1) mRNA is directly translated.
2) Episomal DNA circles form.
3) Integration into the genome.
They have some disadvantages.
• Integration is random.
• Expression under control of highly active LTRs (long terminal repeats) which are transcribed into DNA by retroviruses.
• Cancer risk if inserted next to a proto-oncogene.
• Clinical trials halted e.g. SCID.
What are lentiviruses?
A lentivirus is any retroviruses which has a delay in symptom onset.
• Based on heavily modified HIV.
• Infect primary cells or cell lines with up to 100% efficiency.
• Uses envelope protein to bind to the target cell.
• LV RNA is copied into DNA with RT.
• DNA pre-integration complex enters the nucleus and integrates into the target cell’s chromosomal DNA.
• Gene delivery is stable as GOI is integrated into the chromosome and replicated with gDNA.
• Safe harbour technology to try and ensure GOI is expressed and to prevent cancer risk.
• LVs can infect and integrate into non-dividing cells.
What are induced pluripotent stem cells?
iSPCs reset mature cells back to an early embryonic state very similar to embryonic stem cells.
• Uses panel of genes: Oct4, Sox2, cMyc ad Klf4. Nanog and Lin28 improve efficiency. These are called Yamanaka factors.
• Low efficiency. Incomplete programming. Genomic insertion. Tumorigenesis.
• Can re-differentiate them into new cell types.
