Introduction: Microinjections & Viral Methods Flashcards
What is transgenesis:
Transfer of cloned genetic material into living cells to creat transgenic animals…
For the purpose of understanding and manipulating gene function
What is functional genomics?
Understanding the physiological functions of genes
5 benefits of using germline transgenesis?
It doesn’t carry the same limitations faced by classical genetics and in vitro techniques, such as…
1) Chances of identifying a mutation within the system of interest is very low and breeding of the molecular characterisation of mutation is hard
2) The difficulty in finding cell line (e.g. neurons don’t divide, so can’t get a true neuronal cell line) … therefore complexity of organisms can’t be duplicated
Other benefits:
3) maintains complexity of organism
4) gene sequence can be characterised without prior knowledge of its function
5) can perform reverse genetics
Transgenesis and the germ line… what are the important stages?
It’s important to insert DNA during:
The zygote stage-
Consists of male and female pronucleus - male pronucleus is larger due to the process of taking out protamine (sperm histones), thus many enzymes are acting in this site.
Due to the size, the male pronucleus is targeted in the ‘microinjection of fertilised one-cell eggs’.
The morula stage -
Consists of 16 blastomeres that are pluripotent
DNA insertion at these stages are important for ensuring gene modifications in subsequent generations: As totipotent cells are required to ensure modification is present in whole organism
What effects correct transgene expression?
What about it’s design(3)?
Transgene expression is driven by the cis-acting elements placed in sequence
Things to consider when designing transgene;
1) Must be able to differentiate between transgene and endogenous gene (e.g. tags)
2) Validate the construct - through sequencing
3) Injecting of fragments (Transgene is cut out of plasmid and inserted into host), because:
- Circular DNA doesn’t integrate well
- mammalian cells likely to reject foreign bacterial DNA (plasmid sequences could cause inhibition of the expression of the gene)
How does positioning of inserted transgene effect expression?
1) Repression: Transgene may have randomly integrated in region that is transciptionally inactive (aka inert)
2) endoplasmic expression: Transgene may have integrated close to an enhancer of another gene that is not our gene of interest
What is a retrovirus?
What is the retroviral life cycle?
Structure…
1Capsid
-Envelop (lipid bilayer)
-Virally engineered enzymes: integrase, protease, reverse transcriptase
- RNA genome: 2 copies of single stranded RNA 9,000bp.
RNA structure: contains long terminal repeat (LTR) domain. This domain regulated transformation into DNA and expression.
Has R repeat sequence and unique U3 and U5 sequences.
Retroviral life cycle is for the purpose of mass producing modified viruses
1) Envelope locates to hosts membrane, where it fuses.
2) Capsid is released into membrane and opens up
3) A DNA strand is made from RNA by reverse transcriptase
4) hosts enzyme turns ssDNA into double stranded DNA (dsDNA)
5) viral genome becomes integrated into host genome - ‘provirus’
6) LTR (long terminal repeats) drives expression of products
7) transcription and translation occur, resulting in: envelope proteins, capsid and reverse transcriptase products …. Note: RNA structure has domains for each
What is provirus synthesis?
Steps.
Is the process of synthesising a DNA strand from RNA, by virtue of tRNA which acts as primer
1) endogenous tRNA hybridises to primer binding site (PBS)
2) tRNA primer is extended: creates cDNA of genomic 5’ end
3) RNase H removes hybridised RNA
4) first jump: R sequence within cDNA hybridises to R sequence of RNA in 3’ end
5) strand extension by reverse transcriptase
6) most of RNA is removed - and 3’ of second DNA strand is synthesised
7) RNA and tRNA are removed
8) second jump: PBS sequence on 2nd strand DNA hybridises to PBS sequence in 3’- 5’ strand - both strands are completed
What is a recombinant retrovirus?
Method, advantages and disadvantages…
Concept: Envelope, Capsid and Reverse transcriptase sequences are taken out of gene
Because we don’t want the animal producing those,
The viral particles are solely for the purpose of transporting our gene of interest into the host
So steps entail making packaged product in culture before transfecting animal
1) Make cell that produces gag,pol,env
2) Transfect cell with viral vector
3) Cell now produces packaging proteins and gene of interest
4) Transfection: injection into morula and implantation in pseudopregnant female
Disadvantages:
- small insert size (5kbp)
- potentially biologically hazardous: if animal were to be infected by wild type retrovirus. Would result in suicide viruses
- only infects dividing cells
- powerful LTR: could insert next to oncogene leading to the expression of cancer
- developmental silencing (e.g. methylation)
- have to reclone transgene
Advantages
- efficient integration : high infectivity: high probability of hitting a gene.
- thus, ease of cloning mutated gene
What is a lentivirus…
method, advantages and disadvantages?
Difference - this is a fertilised egg infection (not at morula stage like retrovirus)
1) Components of…
- viral vector (only has LTR sites)
- capsid and enzyme packaging components
- envelope packaging components
… are transfected into a producer cell
2) mature virions from producer cell are harvested
3) Injection in perivitelline space
Disadvantages: (same as recombinant retrovirus)
- have to reclone transgene
- powerful LTR (oncogene expression- cancer)
- potential biological hazard (suicide virus)
Advantages: (make up for some of the recombinant retrovirus disadvantages)
- larger insert (8kbp)
- infects both non-dividing and dividing cells
- no developmental silencing
Microinjection protocol?
Incl. steps and integration info
Microinjection in one-cell eggs:
Manipulation of fertilised egg
1) Mate a male stud and female mouse..
- natural: female mouse is in estrus (can only obtain up to 10 eggs - inefficient)
- super ovulated: Can obtain 80-100 eggs
Day 1: give immature female mouse follicle stimulating hormone (FSH)
Day 3: inject with human chorionic gonadotropin (hCG) - mimics LH
3) look for vaginal plug, isolate eggs
4) Microinject egg with transgenic DNA : hold egg in place by suction and microinject male pronucleus
5) Implant eggs into a pseudopregnant female mouse - mated with sterile male - burst of prolactin release
6) 3 weeks later: pups born
- only ~25% will have transgene
7) perform PCR or southern blot to see which ones carry the gene
Integration is random and occurs once per nucleus. One integration contains 1 - 1,000 copies of transgene
