Practical Stuff Flashcards
What do expression plasmids need extra from cling vectors?
Both need an ori and selectable marker eg AmpR gene
Expression need MCS and an expression marker eg. LacZ gene- beta-galactosidase metabolises X-gal producing a blue product- it will be interrupted if the gene has been successfully introduced- no blue
What are the three most important signals for expressing genes in bacteria?
Promoter- determines the rate that mRNA is synthesised. Can use inducible or repressible promoters.
Terminator
Ribosome binding site- the Shine-Delgarno sequence and Kozak sequence
How could you introduce DNA into the bacteria?
Micro injection
Viral transfection
Heat shock
Electroporation
What are he limitations of using E. coli ?
Limited post translational modification eg. No glycosylation
Improper folding
Degradation
Lack of ability to perform intron-exon splicing
Codon bias- difficulties in host tRNA translating the mRNA
Foreign genes may contain bacterial termination signals
What are the pros and cons of using yeast in cloning?
Is eukaryotic so will be more homologous than E. coli- post translational modification and protein degradation
High yeilds
But still codon bias and proteins are often hyper glycosylated
What are the pros and cons of using mammalian cells?
Difficult and costly and cells usually require a surface to grow on rather than a suspension
If gene is over expressed it can have deleterious effect on the cell so it will be down regulated
But the protein can be accurately post translationally modified and spliced etc.
Describe vectors
A vehicle used to transfer genetic material to a target cell
It needs to be capable of independent replication within a host and of carrying a fragment of foreign DNA
Eg. Plasmids- 10kb, bacteriophages lambda phage-23kb, cosmids 30-44kb, YACS 0.2-2Mb, BACS 300kb
What is gene editing?
Enables manipulation of virtually any gene in a diverse range cell types and organisms
Based in the use of engineered nucleases
Outline the methods of genome editing
Zinc-finger nucleases (ZFNs)- zinc fingers recognise a codon- attached to Fok1 which cleaves in pairs- two different series of zinc-fingers are required on either side of the cleavage site
Transcription Activator-Like Effector Nucleases (TALENS)- also uses Fok1 but uses repeat variable diresidues (RVD) to recognise individual nucleotides- one either side of the cleavage site
Clustered Regularly Interspaced Pandendromic Repeat (CRISPR)- CRISPR Associated Protein 9 (Cas9)- Cas9 is the nuclease, requires tracrRNA and crRNA- cleaves at the end of the non-complementary strand to the guide sequence on the crRNA
Give an overview of using ZFNs in gene editing
Sequence specificity- long target site
Modular customisable DNA binding domains
Limitations- poor targeting density
Target site limitations- G-rich and methylation sensitive
Not all newly synthesised ZFNs are able to cleave chromosomal DNA
Low success rate
Has a high amount of off target effects
Variable cytotoxicity
Costly and difficult to produce and deliver
Give an overview of using TALENS for gene editing
Can be used to recognise small sequences High success rate Low off target effects Less costly to produce Limitations- large constructs Highly homologous- self-recombination Target site limitations- 5' thymine and methylation sensitive More difficult to deliver
Give an overview of using CRISPR-Cas9 for gene editing
Has a high success rate Capable of multiplexing Low cytotoxicity Affordable Easy to modify Easy to deliver Limitations- restriction site limitations- end with an NGG or NAG Has variable off target effects Big contruct
How could you deliver reprogrammable nucleases?
Via plasmid DNA In vitro transcribed mRNA Non-integrating viral vectors Purified protein ➡️ Electroporation Liposome transfection Micro injection
What are the applications of genome editing?
Pre-clinical
-Targeting non-coding regions
-Creation of genetic variation and study their effects
-Generation of knock-out and knock-in models by direct injection into embryos
-Create an isogenic cell line to model human disease
Biotechnology
-GM crops and livestock
-Production of therapeutic proteins in cultured cell lines
Therapeutics
-Treatment of HIV in humans
-Treatment of haemophilia B in mice
-Gene correction and addition in patient-derived pluripotent stem cells for patients with genetic diseases
What cell activity does genome editing rely on?
Double strand break repair
NHEJ repair can lead to deletions or inserting that can knock out a gene
Insertion of donor DNA, single nucleotides or tags
Describe how HIV can be treated with genome editing
People homozygous for the CCR5delta32 mutation of the CD4 T cell receptor are resistant to HIV
ZFN/NHEJ knock out of CCR5 in humanised mouse model of HIV showed reduced viral load and improved CD4 T cell counts
How could you correct the gene dosage for trisomy 21 using gene editing?
Cover the chromosome with Xist RNA- similar to how the extra X chromosome is in women
Using ZFN to insert Xist cDNA
Creates a Chr21 Barr body
Describe a safety mechanism for genome editing
Self-cleaving delivery vehicle
Plasmid also contains a cleavage site for the nuclease
What makes a good model organism?
Developmental anatomy
Size and complexity of housing required
Generation time
Ease of study- genome sequence, size, mammal, non-mammal embryo models
Transparency
Organismal complexity or simplicity
Ethical and legal considerations- reduction, refinement, replacement
Define the three Rs of animal research
Replacement- methods to avoid or replace the use of animals
Eg. Human volunteers, tissues and cells
Mathematical models
Established cell lines
Immature forms of vertebrates or invertebrates
Reduction- methods which minimise the number of animals used per experiment
Eg. Improved experimental design and statistical analysis, sharing data and resources
Use if imaging technologies to enable longitudinal studies in the same animal
Refinement- methods to minimise suffering and improve animal welfare
Eg. Appropriate anaesthetics and analgesics, avoiding stress
Using appropriate housing that allows the expression if species specific behaviours
Why are Drosophila melanogaster a good model?
65% of known human disease loci have clear homologues in Drosophila
More than 100 yrs of genetic research has generated powerful tools- first genome to be sequenced and annotated
Excellent model for discovering gene function and cellular pathways
Complex nervous system and behaviours
How would you analyse gene function in Drosophila?
- Make a mutant (loss of function)- imprecise excision of P-elements, homologous recombination
- Overexpress the gene (gain of function, eg. The Gal4-UAS system
- Knockdown gene excited expression (RNAi) eg. UAS dsRNA X Gal4 driver leads to tissue specific knockdown
What are the most common vertebrate models of human development?
Clawed frog- Xenopus laevis/tropicalis Chick- Gallus gallus Mouse- Mus musculus Rat- Rattus norvegicus Zebrafish- Danio rerio Medaka- Oryzias latipes
Describe the usefulness of using zebrafish for human models
Vertebrate Transparent Large numbers Ease of micro manipulation, grafting, filming, phenotype screening Forward genetics Fast 'gene knock down' Knock out with ZFN and Crispr Cas9 Genomic resources
How would you generate animal models if disease?
Spontaneous mutants- hard to find
Eg. Haemophiliac dog, loss of function IX
Watanabe heritable hyperlipaemic rabbit- familial hypercholesteraemia
Induced mutagenesis- forward genetics
-chemical treatment or irradiation of sperm
But may soon us random an screening laborious
Transgenic models- reverse genetics
Which diseases are zebrafish models being developed for?
Cardiovascular Cancers Neurodegeneration Congenital dismorphology- (holoprosencephaly) Behaviour Rare genetic disorders Regeneration models Methods of analysis include- genetic analysis, visualisation, drug high throughput screening
Describe the clawed frog as an animal model
Tetrapods
Big embryos available in large numbers
Excellent for micro manipulation, grafting and culture studies
Excellent for overexpression studies
Reliable fate map
Knock downs possible with anti-sense oligonucleotides
Describe chicks as a model
Amniotes
Excellent for micromanipulation and tissue grafting
Good for overexpression studies
Describe the mouse as an experimental system
Genome- 99% of its ~25000 genes have a human counterpart
Life cycle- 4 day oestrus, 20 day gestation, 4-8 pups per litter, 2-8 litters per female, 7 weeks to sexual maturity, 2-3 yr lifespan
Reverse genetics- knockouts transgenics, conditional expression, inducible expression, retroviral vectors, siRNA
Strains- inbred, out red, recombinant inbred, consomic, fluorescent
Assisted reproduction- cryopreservation, embryo rederivation, in vitro fertilisation, intracytoplasmic sperm injections, cloning
Tools- genome sequencing, embryonic stem cells, expression arrays, gene trap libraries, insertional vector libraries, BAC libraries
How can you create a transgenic mouse?
- Build transgene construct
- Introduce into cell (zygote or ESC)
- Integrate DNA into genome
- Select transgene carriers- embryo➡️mosaic or ESC➡️chimaera
- Implant into foster mother
- Breed chimaeric offspring to select for heterozygotes
- Make homozygous by sister crossing
Describe the transformation of cells/zygotes
Transduction- virally mediated gene transfer- AAV naturally integrates into the genome
Transfection- chemical or electric mediated mediated gene transfer- CaPO4, lipid complexes, Electroporation
Direct transfer- micro injection, particle bombardment
Describe the implantation of the transgenic zygote/embryo
Needle is used to introduce zygotes into the uterus of a pseudopregnant recipient female
(Copulated with vasectomised male)
What is the advantage of using homologous recombination for transgene integration?
Can target site
Replace genes
Knock out
Describe the positive and negative selection of targeted ES cells
Some cells will be non-recombinant, some recombinant with random inserts and some recombinant a with gene target insertion
- Selection for antibiotic resistance marker gene eg. Neor- neomycin removes non recombinants
- Selection for gene that should have been removed by targeted homologous recombination eg. TK- ganciclovir removes the random insert recombinants
What is a conditional knock out?
The target gene can be eliminated in a single organ
Cre/IoxP mediated gene deletion system
Cre recombinase recognises IoxP flanked sequences
It is only expressed if there is a tissue specific promoter
What are the pros and cons of mouse embryo manipulation?
Allows genetic manipulation of embryo possible
Early embryos can be split to yeild two twins
Two morulas can be combined to form a chimera
Cells from an embryo can be injected into another blastocyst to form a chimera
However embryonic manipulation is difficult inside the mother
Extra-uterine embryo culture is difficult, need to cut open the uterus- ethics
What are the similarities and differences between the human and mouse brain?
90% identical genes for brain development and function
Tool for the study of human mantas disorders, memory, intellegence
However the mouse brain has a smooth surface and less memory, whereas the human brain has more convulsions and higher order memory
Mouse- 70% neurons, 30% glia
Human- 30% neurons, 70% glia
What are the advantages and disadvantages of using the mouse brain as a model?
Shares may features like anxiety, hunger, circadian rhythm, memory
Robust phenotypes in mouse models
Treatments of autism spectrum
Effects of alcoholism
However animals lack self-consciousness, self-reflection and consideration
Lab vs natural environments
Some behavioural disorders such as depression, low self esteem and suicidal are inaccessible
