When building a transgene, what do eukaryotic cells need?
1) A promoter that will drive expression in the tissue that you require
2) An open reading frame encoding the gene you want to express
3) Sequences that ensure correct mRNA processing e.g polyadenylation signal AAUAAA
Steps of building a trasngene:
1. Acquire complimentary DNA - this is mRNA that is reverse transcribed - the effect of this is that you get an open reading frame (with no introns in it)
2. Incorporate a promoter - this is where transcription machinery binds
3. Incorporate regulatory elements - these are small DNA sequences that are known to be bound by transcription factors that activate or repress transcription
2 and 3 are often taken from other genes in the tissue
4. add a polyadenylation signal
5. Add an intron (mRNAs are processed better if they have an intron - tends to be an intron from beta globin)
What are methods of introducing transgenes into an animal?
Direct Injection of DNA into cells
Using a micropipette
We can directly inject DNA into a fertilised egg
There is a period of time where fertilisation of an egg has happened but the pronucleus of the sperm has not yet fused with the pronucleus of the egg. We can inject the DNA into the pro-nucleus of the sperm.
DNA repair mechanisms recognise this free DNA in the cell and try to do something with it - may accidentally introduce breaks in host DNA and ligate transgene.
Incubate cells in culture medium containing DNA and a chemical that wraps the DNA up and is either endocytosed or that diffuses through the cell membrane
Expose cells to DNA and give them an electric shock to force DNA into the cells
Expose cells to viruses carrying transgene DNA that will infect cells
How are transgenes often integrated as a result of DNA repair enzymes?
They are often integrated as concatamers - tie up lots of copies of the transgene in a line which is then integrated - you lose control of gene dosage. Too many genes in a sequence may be turned off like a viral sequence.
When the transgenic animal is made, what are the potantial reasons that gene expression is not as you had expected?
Weak promoter / Insufficient regulatory elements
Copy number (too many or too little)
Position effects (site of integration) - it could end up in a centromere and silenced, or it could enter near another gene - might adopt the expression pattern of this gene.
Genetic background (farm animals, hence Dolly) - not really the case with mice. Inbred strains are identical.
Very big transgenes (>1 Mb), which put the promoter of the transgene in its normal chromosomal context, usually work best
How can viruses be used to insert a transgene?
Retroviruses infect host cells and then reverse transcribe RNA into DNA, this DNA is then integrated into the host genome
What are the components of this 'typical retrovirus'?
gag ⟶ encodes proteins of nucleoprotein core of virion
pol ⟶ encodes reverse transcriptase, integrase etc functions
env ⟶ encodes surface protein components of virion
Y ⟶ packaging signal - any RNA with this signal will be incorporated into the viral particle. Without this sequence the particle is not packaged.
There will also be flanking sequences
Viruses aren't genereally speaking big enough to carry their own genome plus the transgene, how do we get around this?
We have to remove the host genome and replace it with our genome.
We surround our transgene with viral flanking sequences as well as the packaging signal. However this combination won't do anything without the viral genome, but the viral DNA has to be somewhere else within the cells that are making the virus.
We get eukaryotic cells from tissue culture, we place inside them (by injection, transfection etc) is our transgene (with our flanking sequence and packaging signal) alongside a copy of the viral genome without the packaging sequence - both these things are active producing RNA - but the components of the virus (gag, pol, env) will be being made by the retrovirus but the retroviral RNA cannot be packaged. So the whole virus will be assembled but the only thing we can put into the virus is out transgene with the packaging sequence.
The cells nowadays already have the gag, pol and env inside them.
Once we have these viruses that contain our transgene, what can they be used to infect?
1-2 cell embryos - although these transgenes are often silenced
Infect cells in vitro in particular, eyes, limbs, etc of later embryos
Viruese are not often used to create transgenic animals in which every cell contains the transgene, they are more often used to introduce a gene into bits of the mid-gestation embryo to create a mosaic.
Why are viral vectors often a very poor means of creating transgenic mice?
As a combative means to defending against viruses the inserted DNA is often silenced, long-term expression is a problem.
They may only work in cells that are dividing
Side effects - recombination could lead to infective viruses.
What type of virus are the retroviruses used?
They come under the heading lentiviruses. The most commonly used versions are derived from HIV but engineered to ensure that no replication competent virus can be produced.
They are effective at infecting dividing and non-dividing cells, they also have a stable integration and are capable of long-term expression of a transgene.
For what purpose is viral mediated transgenesis popular?
Viral mediated transgenesis is applicable to all organisms and is therefore popular with people who work on animals where other methods of transgenesis are not yet mainstream e.g. chicken
Viruses also good at integrating inhibitory shDNA for the purposes of gene silencing
What are the two main downfalls of direct injection of DNA into a host nucleus?
Uncontrolled integration site - may cause mutations.
Also expression is unpredictable as the transgenes are often integrated as concatemer