paper 2 Flashcards

Question

what is the most popular FRET pair?

Answer 1

cyan FP-yellow FP a few CFPs have high quantum yield but also have fast photobleaching and photoconversion of YFPs into CFP-like FPs

Answer 2

for levels of molecules like Ca, PKA and signalling molecules - reliant on conformational change induced by binding of signalling molecule - brings together 2 fluorophores and increases FRET efficiency

Answer 3

**Sasmal 2020**: FRET assays to assess TCR-pMHC binding * labelling with Cy5 and Cy3 (acceptor and donor) * FRET efficiency = indicator of compactness of TCR-pMHC bond * showed different TCR-pMHC bond compactness correlated to amplitude of T-cel Ca release in vitro * proposed TCR discriminates between similar peptides presented on MHCs by different compactness of bonds

Answer 4

* FRET is limited by need to genetically encode specific proteins * also genetically conjugating proteins to a fluorophore may alter cellular distribution of protein * IHC must go alongside

Answer 5

widely used technique to track migration, proliferation and differentiation of specific cells in vivo usually performed w Cre-LoxP * Cre recombinase expressed under control of tissue or cell specific promoter in a mouse line * this line is crossed with second mouse line in which a reporter (usually tdTomato) is flanked by floxed STOP sequence * in animals expressing both, Cre specifically activates reporter in cells expressing the promoter by excising the STOP sequence

Answer 6

enzyme reporter like LacZ gene (encodes b-galactosidase) produces intense blue signal when incubated with substrate analogue x-gal - colorimetric assay, can be permanently fixed by enzyme activity cannot be viewed in living tissue

Answer 7

genetic cell-labelling technique where hundreds of different hues can be generated by stochastic and combinatorial expression of a few spectrally distinct fluorescent proteins i.e. red, green and blue can combine to generate all colours, 3 or 4 GFPs can be expressed at different ratios in each cell to create colour combos unique to each Brainbow-expressing cell

Answer 8

DNA excision-based Brainbow (1.0): 3 separate FPs are arranged sequentially in transgene with 2 pairs of Cre recombinase recognition sites (Lox) flanking 1st and 2nd FPs * 2 pairs of lox sites can only be recognised by Cre in identical pairs * Livet et al 2007 * crossed 4 Cre lines known to trigger expression in different parts of the brain * authors showed 99% of randomly selected neurons were sufficiently different to distinguish under confocal microscopy

Answer 9

use of 1ry antibodies to target antigen and 2ry antibodies to target 1ry * laborious and has risk of non-specific binding by 2ry * also cells need to be dead and fixed unlike GFP-conjugated proteins * need to be permeabilised limiting potential analysis of membrane-bound proteins

Answer 10

* FITC (fluorescein isothiocyanate) * TRITC (tetramethylrhodamine ...) derivatives of isothiocyanate with modifications that allow them to be conjugated to specific proteins`

Answer 11

* single cells are analysed in individual droplets + analysed at high speed by laser illumination * multiple lasers @ specific frequencies excite fluorophores, which emit light at selected wavelength

Answer 12

* mammary-specific polyomavirus middle T antigen OVX mouse (MMTV-PyMT) = most commonly used genetic engineered mouse * MMTV-Myc: prone to mammary tumour * E-mu-Myc: prone to B cell lymphoma * Tp53 KO * Capecchi, Smithies + Evans received Nobel Prize in 2007 for developing tech for introducing specific mutations into mouse germline (KO or KI)

Answer 13

* rodents are small, easy to maintain, have a short life cycle + gestation periods * mouse + rat genomes sequenced in 2002 + 2004 * many similarities to humans in terms of anatomy + physiology * 95% of genes are shared by mice, rats and humans

Answer 14

* hereditary deafness: difficult to study in humans due to lack of large families + genetic heterogeneity, can control environmental effects wholely in mice *

Answer 15

* cardiovascular research: larger size facilitates surgical procedures * behavioural studies: much more social than mice, better replicate humans * toxicology

Answer 16

fragile X syndrome * expansion of 3 base pair sequence in FMR1 is responsible for fragile X syndrome (most common cause of inherented intellectual disability) * expansion leads to methylation of FMR1 gene, shutting it down an stopping expression * can cause autism * when FMR1 KO'd in mice, become more social, when KO'd in rats, become less engaged in social play + emit fewer vocalisations, more closely mimicking social behaviour symptoms in humans * also have compulsive chewing behaviour, therefore more appropriate model

Answer 17

* depends on which species more closely replicates symptoms + disease process of humans * saves a billion money from testing drugs + therapies, if they fail at later stages its even more expensive * results in animals do not always reflect human outcomes * of 5k that enter development, 250 make it to preclinical, 5 to human and 1 to the market: time and money loss HUGE * establishing more specific + sensitive preclinical trial paradigm based on better animal models will reduce drug development costs + reduce risk to human subjects

Answer 18

* **Engel 1997**: mice lacking T/B cells: Rag2-/-, SCID mice show heightened suceptibility to tumour induction by carcinogens MCA or DMBA + TPA * identified cell types (T, NKT, NK cells) + effector molecules (perforin, TRAIL, IFN-y, type I IFNs) involved in tumour elimination + immunoediting, **using mice lacking all of these genes individually**, each mouse demonstrates enhacned susceptibility to **fibrosarcoma induction**. suggests these immune cells use these pathways to suppress in vivo tumour growth

Answer 19

oncolytic viruses: studied in mouse models for ability to replicate in and lyse tumour cells selectively * mice been used to observe release of tumour antigens, danger signals, induce immune responses and make tumours 'hot'

Answer 20

1. syngeneic tumour cells 2. genetically engineered 3. patient-derived xenograft 4. humanised PDX

Answer 21

Adm and Vegfr3-CreER(T2); Cx43(fl/fl) mice looking at cardioprotective peptide, oedema and MI related to Cx43

paper 2 Flashcards

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