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
