Lecture 8 Flashcards
Descrbe genome editting vis THE CRISPR/Cas9 system
- Clustered regulatory interspaced short palindromic repeats (CRISPR) are loci that contain multiple short direct repeats and provide acquired immunity to bacteria.
- CRISPR RNA base pairs with trans-activating chimeric RNA (tracrRNA) to form a two-RNA structure that guides Cas9 endonuclease to complementary DNA sites for cleavage of specific DNA sites.
- Cas9 serves as the RNA- guided DNA endonuclease that cleaves DNA when crRNA-tracrRNA complex target a recognition site with genome structure.
- A protospacer adjacent motif (PAM) is a 2-6 base pair DNA located immediately downstream of the DNA sequence targeted by Cas9 nuclease. This is needed in order for Cas9 to cleave the targeted DNA sequence.
Cas9 also cleave few base pairs upstream of PAM sequence and a common PAM sequence is NNG in which N can be any bases followed by two guanine bases.
What are the genome editing outcome from CRISPR/CAS9 system?
- Gene addition: Cleaving particular site of genome and then add donor transgene by homology directed repair.
- Gene disruption via small insertions/deletions: Cleaving particular site of genome and then insert double-stranded oligonucleotides (up to 14KB) into that particular site.
- Gene deletion: Simultaneous cleavage of two different genome sites by two CAS9 endonuclease to remove a gene. Can be used to remove gene coding for receptor responsible for disease to manage disease symptom
Explain conditional knockout
Conditional knock-outs involve knocking out gene encoding receptor in a particular cells e.g. endothelial cells in cardiovascular systems as drug target of interest is endothelial cells receptor.
Explain what happen when cros breeding CRE mouse and LoxP mouse?
○ Cre LoxP mouse that have endothelial cells expressing Cre recombinase and Cre remove target genes between two LoxP sites, allowing the reporter gene that encode green fluorescent protein to be transcribed instead.
○ Cre LoxP mouse that have endothelial cells that don’t express Cre recombinase and the target genes is expressed instead of reporter gene as the stop codon between the two gene stop the expression of reporter gene.
- The target gene might encode beta adrenoreceptor in endothelial cells. In Cre LoxP mouse, the endothelial cells will be stained with green fluorescent protein as cre recombinase remove beta adrenoreceptor gene from endothelial cells. But other cells will not be stained with green fluorescent protein and their beta adrenoreceptor will still be expressed as it doesn’t have endothelial cell specific promoter.
Describe the phasge display approaches to develop antibodies
- Gene of interest cloned into gene 3 protein of phage which encode for expression of protein (antibody or peptide) on bacterial phage surface.
- Then, can generate a huge library of all potential genes encoding for antibodies of interest.
- Use phage displaying the protein of interest in library of genes to try and identify particular phages that express good antibodies for antigen of interest.
- Create a screening plate that have desired immobilized antigen on plate surface and run phage across plate.
- Some displayed antibodies will bind to immobilized antigen, causing the phage to stick to plate.
- The plate is washed to remove unbound antibodies of phage and the remaining bounded phage is eluted.
- Then, infect more E-coli bacteria with bounded phage to create more phage containing the good antibodies, amplifying the number of antibodies which are enriched for binding to antigen of interest.
This reduce down the library to a few high affinity binding antibodies which can be further isolated to get one antibody of interest.
What is the phage approach used for?
It is used to identify nanobodies (single domain antibody fragment)
- Camelids (camels, llamas, alpacas) express heavy chain antibodies which has a variable region that has antibody function.
- Shark express distinct heavy chain antibodies which is similar to camelids heavy chain antibodies
- Isolate variable region (nanobodies) from llama heavy chain. The nanobody is small (15kDa).
- Use phage display approach:
○ Llama immunised with antigen of interest
○ B cells used to isolate gene encoding single heavy chain antibodies with variable region
○ Insert gene on phage to express heavy chain antibodies on phage surface
Screening of phage to identify nanobody that bind well to antigen of interest.
What happen when P2Y12 receptor are activated?
Activation of P2Y1 by ADP, a GQ coupled receptor leads to increase in intracellular calcium in which leads to changes in platelet shape, platelet aggregation and platelet secretion.
What happen when P2Y2 receptor is activated?
Activation of P2Y12, a Gi coupled receptor prevent the activation of adenlyl cyclase thus reducing cyclic AMP levels and reduce protein kinase A activation. Instead it stimulate PI3K, rap proteins and AKT phosphorylation, leading to signal amplification to amplify change in platelet shape, platelet aggregation and secretion. It also amplify signals from P2Y1 receptors.
How can ATP can cancer metastasis?
- Tumour cells can bind to and activate circulating platelet, leading to ATP release.
ATP bind to P2Y12 receptor on endothelial cells to cause endothelial cells retraction to open up gaps for tumour cells to leave vessel lumen and infiltrate surrounding tissues.
How can cancer metastais be prevented?
- Experiment to validate role of ATP in cancer metastasis:
○ Apyrase catalyse the hydrolysis of ATP and ADP to ADP and pyrophosphate.
If treat cells with apyrase, it can prevent platelet-dependent facilitation of tumour cell migration.
describe how clopidogrel inhibit P2Y12
what receptor is expressed predominantly in platelet
what is the competitive antagonist of ADP induced platelet aggregation
give an example of allosteric antagonist of P2Y12 receptor
