Getting DNA into cells and DNA analysis

Why do we deliver DNA into cells?

• Study gene function and regulation

• Produce proteins

• Generate GM organisms

• Develop gene therapies

What is the main problem when getting DNA into cells?

DNA must cross the cell membrane and nuclear envelope

What are the two main method of getting DNA into bacteria?

• Cosmids

• Physical methods

What are the physical methods of getting DNA into bacteria?

Chemical transformation and electroporation

What is a cosmid?

Phage mediated shells can be generated by in vitro packaging

How do we know bacteria can take up DNA without any intervention?

Fred griffins experiment

• Heat kill smooth strain and inject into mice - lives

• Rough strain in (live) and inject - lives

• If do both, mice dies - smooth strains DNA was transferred into the rough strains

Describe the process of chemical transformation

• Have natural competence - they have adhesion zones on their cell membrane that can take up DNA

• Lipid bilayer has a negative charge, and so does DNA - there are electrostatic repulsions so not easy to get DNA in

• Calcium chloride is added to neutralise the DNA and membrsned negative charge - reduce the electrostatic repulsions

• Cooling down the cells reduce the movement of membrane components, allowing DNA to get in (membrane is more static)

• Cells are heted at 42 - this sets up a temperature gradient that works to ‘suck’ the DNA in, temperature gradient derived current drives the DNA in

Describe the recovery after chemical transformation

• Added LB and growth without antibiotics

• Selection markers in the bacteria (antibiotic resistance gene) has time to be expressed

• Let them grow without any selection markers

Describe the plating after chemical transformation

• Plate in the selective conditions

• Only those that have taken up the plasmid can grow on the selective plate

Describe the process of electroporation

• Need a specialised kit - electroporator - and specialised cuvettes with metal electrodes

• Do at 0C - minimize hear damage

• Can make elctrocompetent by washing to remove salts

• Bacteria resuspended in water and DNA in water is put into cells

• High localised electric current makes the transient pores in the membrane

• -vely charged DNA passes through in an electrophoresis type effect

Describe the use of bacteriophage infection for transfection

• Bacteriophages can be modified to allow delivery

• Typically use lambda phage

• Cosmid vectors - modified bacteriophage like genome, clone GOI in. In vitro assemble into empty shells of viral capsule. Mix with bacteria and DNA is put in

• Not used routinely - only used when you have very difficult to transform cells → some bacteria are transform resistant

Describe the types of selection that can be used to identify transformed cells

Antibiotic resistance markers

• Only cells with the plasmid can survive and propagate on selective medium

Blue white screening

• Uses the disruption of the LacZ gene

• Different colours allow identification of those that have taken up the plasmid

Why can’t even selection markers be 100% certain those cells have been transformed?

not all growing on the plate might be the correct clone - even if they have passes the selection processes → they may have random mutations

What are the methods to verify cloning?

• Colony PCR

• Restriction digest analysis

• DNA sequencing

Describe the use of colony PCR to verify cloning

• Select colonies, make a suspension in water and use as a template for a PCR reaction

• Have specific primer pairs - use primers that match the gene of interest - if the primers have that piece of DNA you can see

• Screen for presence of the gene of interest

• Can make sure you position the primers just outside of regions flanking the GOI - make sure its the right size

• Can also check the orientation using specific primers that give you a readout of the orientation

Describe the use of restriction digest analysis to verify cloning

• Once you have the colonies, grow them up and do a miniprep

• Test the plasmid by restriction digestion

• Can choose restriction enzymes that release the gene of interest → have RE sites at either end of the GOI → Can then do an electrophoresis to check for the correct banding

• Cut with the same RE at multiple sites, check banding is right to see if in the right plasmid

• Can chose enzyme that cuts outside and inside - select for the orientation as different orientations will give different sized bands

Describe the use of DNA sequencing to confirm cloning

Can use to confirm the exact sqeueqnce and reading frame

Why may mammalian cells be transformed?

• Understand cell biology

• Manufacture proteins

• Test drugs and therapies

• Regernative medicine

What are primary cells?

• Not immortal - only grow and divide about 50 times and then they die out (Hayflick limit) → finite growth

• Can express telomerase in the cells and they become immortal

What are immortalised/transformed cells?

• Primary cells with telomerase being expressed - do not have the hayflick limit

• Could also use cancer cells - as they are also immortal → but these have no contact inhibition like those derived form primary cells

What is the difference between primary and tumour cells?

Primary cells adhere to the surface, touch other cells and stop growing, cancer cells do not do this, they start growing on top of eachother

Why are different conditions required for mammalian transfections?

• Different conditions required as to the type of cell you use

What is the mammalian version of transformation?

Transfection

What is transient transfection and why is it like this?

short tem expression - this is because they exist as their own entities, not integrated into the genome so will be diluted out

What is stable transfection and why is it like this?

long term - they are integrated into the host cell genome, so will replicate with each replication cycle

What must be thought about during transfection?

Efficiency vs toxicity

Cell type sensitivity

What are the physical methods of mammalian transfection?

Electroporation

CaPO4 transfection

Liposomal transfection

Magntofection

Microinjection

Biolistics

Describe CaPO4 transfection

• Mix Ca2+ with DNA, this binds to the surface of the DNA and forms tiny precipitates - these sit in the medium

• Mammalian cells ingest the Ca2+ covered DNA by micropinocytosis → inefficient process

• Cheap but low efficiency

What are the two vectors for miposomal transfection?

Can use liposomes (lipofectamine) or cationic polymers (FuGene)

Describe the use of lipofectamine

• Agent that forms micelles around the DNA

• Micelles fuse with the membrane and form endosomes, this delivers the DNA in the cells → we have made a bilayer around the DNA in vitro

• Membrane fusion

Describe the use of cationic polymers

Positively charged polymers coat the DNA, taken into cell and form endosomes and release DNA

How should you determine the different conditions for cell transfection, and what does this depend on?

Emperically

Need to use different conditions for cells, cell density, transfection reagent and vector/insert combination

Why is liposomal transfection inherently toxic?

Methods are inherently toxic as DNA is delivered into the cytoplasm - thinks it is a viral infection

Describe the process of magnetofection

• DNA is put on magnetic beads,

• Tissue culture dish in the magnetic field, magnetic nanoparticle - vector complex is taken up by endo and pinocytosis

What are the methods for getting DNA into entire organisms?

Microinjection, biolistics

Describe how DNA can be inserted into an entire organism by microinjection using an example?

• Mouse oocyte, use a small pipette to get

• Poke cell with syringe and dispense the DNA into the cell

• Need specialist equipment and people

• Can then use the oocyte to grow transgenic mice

• Can also apply to stem cells in culture

Describe the process of biolistics

• Used in plant research to get DNA into plant cells

• Have small gold particles (microprojectiles) that you coat with DNA of interest, these are suspended in a drop on a macroprojectile. Explosion causes breakage of membrane that releases helium gas that shoots and forces the macroprojectile through the membrane. Macroprojectile hits stopping plate, and micros go onto tissue below, introducing the DNA into the cells

• Can make an entire plant from a single cell

What are biological methods used for transfection?

Agrobacterium

Viruses

Retroviral vectors

How is agrobacterium used for transfection?

• Parisitic bacteria that infect plants

• Ti DNA needed for infection

• Make plasmid with the GOI in E.coli (as it needs the Ti Plasmid DNA containing auxins etc)

• Then transform A tumefeciens with the plasmid and infect cultured plant cells with the engineered agrobacterium. → Ability to integrate their genome into the plants genome - can modify the genome so we can get it in

What viruses can be used for transaction?

Adenoviruses, retroviral vectors, lentiviral vectors

Describe how adenoviruses can be used for transfection

• Most efficient delivery vehicles for DNA into mammalian cells

• Infect almost all cell types - dont have to be actively dividing

Process:

• Clone GOI into shuttle vector recombine with AdEasy plasmid in E.coli.

• Isolate pladmif and linearise

• Liposonman transfection into HEK293 cells

• Infection yields virus that can infect other cell types.

• DNA being delivered is transiently transfected - the adenovirus genome does not integrate into the genome

Describe the use of adeno-associated virus

• Also very efficient ways of getting DNA into hard to transfect cells

• Even better than adenovirus

Describe how retroviral vectors can be used for transfection

• More stable

• RNA genome injected → can be directly translated into the protein of interest, can be reverse transcribed (and will then integrate into the genome)

• Expression is under control of active LTRs → cancer risk if insert next to proto onco gene

Describe the use of lentiviral vectors

• Heavily modified HIV

• RNA genome

• Stable transfection as the RNA integrates into the host genome via reverse transcription, is replicated with gDNA

• Leads to problems

• Related to high oncogenicity - as integration into proto-onco genes can cause cancer

• Can infect and integrate into non dividing cells

How can uptake and expression of DNA be assessed?

• Can use antibiotic resistance selection

• Can also fuse the gene of interest with fluorescent proteins, can observe cells directly - if they have taken up the DNA they will fluoresce → used to test the efficiency of transfection

• Can sort the cells using FACS machine to select/enrich the cells that have taken up the gene of interest (uses GFP)

• Can assess via western blots. ensures the gene of interest is in the cells

What can be used to assess transfection efficiency?

GFP reporters can be used to calculate the transfection efficiency

How can DNA be looked at?

Can visualise by gel electrophoresis and DNA staining in cells

How does the process of a DNA gel work?

• DNA is -vely charged, so will migrate towards a +ve terminal when in an electric field

• Can run through a matrix - will separate according to size → shorter move faster and further, larger dont

Describe the use of agarose gels

• Large fragments

• Depending on how you run the gel, can have an entire chromosome being separated

• Larger pore size

• Running DNA through a matrix in an electric field and separate based on size

→ agarose gels - large pore size, better for large fragments

Describe the use of polyacrylamide gels

• Short fragments

• Allows detection of single nt

→ polyacrylamide - smaller pore size, good for smaller fragments - can give you single base pair resolution

What are the different DNA gels that can be used?

Ethidium bormide

Sybr

GelRed

How does ethidium bromide work?

• Intercalates in the DNA

• Then fluoresces

• Run DNA through the gel, ethidium bromide intercalates and stains the DNA, gel into IV transiliuminator can be used to visualise the bands

How can DNA be labeled inside cells?

DAPI dye

Anti-BrDU

EDU

How does DAPI colour DNA?

intercalates in the DNA in the cell → labels DNA nonspecifically

How can antiBrdU be used to measure DNA?

Can use antibodies anti-BrdU, this are incorporated into the DNA

How is EDU used to measure DNA?

modified uracil has an alkyne group, can use click chemistry to label with fluorophores → can specifically identify cells that are replicating as they incorporate a lot of the fluorophores

How can we look at specific DNA sequences in cells?

DNA probes, FISH

How can DNA probes be used to look at specific DNA sequences?

• Hybridise complementary labeled probes → radiolabelling with phosphate attachments

• Can do in vitro on a gel you have run - southern blot

• Run DNA on a gel, transfer DNA onto a membrane (often a stack of filter paper), sucks liquid through the gel via capillary action → not have DNA on the membrane

• Can denature the DNA (so ssDNA) and add very specific probes - specific sequences → often used to validate gene editing

• Can get two bands as two bands of DNA (if has worked on one and not the other)

• Routine method to validate gene editing

How can FISH be used to look at specific DNA sequences?

• Fluorescent probe

• Denature DNA inside the cell, probe will hybridise in situ and can visualise DNA that has been edited

• Rapidly evolving, more and more sensitive probes and microscopes

• So advanced that you can see very fine structure → recent report - used several probes in a particular locus and detect a loop

What does FISH stand for?

fluorescent insitu hybridisation

Hoe can proteins be visualised using DNA paint?

• Use primary antibodies against a specific protein

• Secondary antibody has DNA tagged to it, these have probes attached that will fluoresce /light up, can be used to show proteins present

What are the ways DNA can be sequenced?

Sanger seqeuncing

NGS

Nanopore seqeuncing

ChiP sequencing

Hi-C

Describe Sanger sequencing

• Works by chain termination

• If you clone something, validate in the lab and then isolate the DNA and do the sequencing

• Can check and verify the mutation has been incorporated

Describe NGS and some of its issues

• Sequencing field is developing at a large pace

• Most commonly used method is Illumina sequencing - sequencing by synthesis allows you to sequence many sequences at the same time - shortens the time to sequence an entire genome

• Issues with illumina sequencing - alignment error derived → repetitive sequences can be difficult to assemble, align and can make it difficult to accurately quantify gene expression levels

Describe the process of nano pore sequencing

• Take DNA of interest from cells, fragment it and then ligate adaptors at the end of the DNA

• Adaptors bind to a specific motor protein (helicase) to pull the fragments through the nanopores

• Helicase bound adaptors are out onto the nanopore sequencing flow cell, this has wells with the capacity to bind to the motor proteins

• Membrane has pore that can bind the long piece of DNA

• Helicase starts to unwind the DNA and pump it through the pore int eh membrane.

• Current flowing, as the DNA passes through it disrupts the electric field, each base has a unique signature of distortion - this is what allows you to sequence → methylated bases also have unique signature distortions - can usr to map modified DNA sequences → efficiently and quick

• 400 bases per second - get lots very quickly, so works fine for repeat sequences

Describe the use of ChIP sequencing

Map where a particular protein of interest is sitting on the DNA - get a footprint of it

Describe the process of ChIP sequencing

• If the protein binds a specific sequence (eg TF), can use cross linkers that non specifically cross link protein to DNA.

• Can then break open the cell, releases the protein bound to gDNA

• chop the gDNA into sequences. Specific antibody binds the POI and pulls down the protein and DNA it is bound to

• Have enriched the protein it was bound to

• Can heat to reverse the crosslink, protein is released and specific DNA is released → this can be sequenced and mapped against the genome to find where the protein was bound

Describe the use of Hi-C

• Allows you to look at 3D organisation in the cell

• Works with the same principle as ChIP

Describe the process of Hi-C

• A protein binds 2 different sequences- forms a loop between different regions of the genome

• This can be cross linked, isolated. Sequences can be ligated together (via RE digestion) , then sequence and map against genome - can then infer interactions between different regions of the genome