Paper 2 Flashcards
MCC mice Tumour development study
Testing efficacy of shRNA knockdown of T-antigens on replicative potential of tumour cells
Subcutaneously injected WaGa MCC cells into 5-week-old NOD/Scid female mice (B and T cell deficient), who after 2 weeks developed visible CD20+ tumours with stromal involvement and MCC-morphology.
Tumour development over two weeks whereas in human disease MCV (viral induced) infection often in childhood and develops over years.
In reality far more tumour heterogeneity is likely
Using doxycycline induced short hairpin RNAs for knockdown - did not assess for off target effects which could further alter survival of cancer cells. Reduce: use control scrambled hairpin RNA.
Using doxycycline inducible models in genetics: always need to control for the body wide effects Dox could be having. Doxycycline antibiotics moa: inhibit bacterial 30s ribosomal subunit.
Reduce: use just dox control without shRNA to control for effect.
Especially given SCID mice – could Dox be affecting disease progression
McNiell et al 2017
Mice are normally very resistant to atherosclerosis, however the homozygous ApoE knockout significantly leads to increased susceptibility for atherosclerosis, with lesions characterized by vascular inflammation and macrophage infiltration as occurs in the human disease.
Used a hCD68GFP transgenic ApoE-/- mice as a macrophage reporter model, and showed abundant GFP expression in arterial lesions.
These ApoE knock-out mice have been widely employed as a platform for the study of various potential mediators or inhibitors of atherosclerosis.
However lipid metabolism is very different between mouse and humans, and mice must undergo a cholesterol-rich diet to even partially mimic atherosclerosis.
Skryabin et al 2020
CRISPR tandem with Cre-Lox to create a conditional knock-in of S100A8 in immune cells.
Frequent unwanted duplications, which could not be picked up by PCR analysis.
Likely unidentified mutations are present in mice that are generated this way, thus making this functional gene studies invalid.
This further implies the need for better understanding of these “revolutionary” gene editing tools, which are often praised for their simplicity.
Mouse model of AD?
There is a strong connection ApoE4 and LOAD risk, with apoE4 carriers accounting for 65-80% of all AD case in most clinical studies, but there is no authentic ortholog for ApoE4 in mouse models.
Transgenic mice expressing human apoE4 isoforms have been developed, but these models have thus far failed to elucidate the mechanism for how these gene manifests in pathology.
Furthermore, diet and exercise are among the leading risk factors, but their long-term effects are very difficult to recreate in animal models.
Preman et al 2021
Transplanting human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors expressing APOE ε3 (E3) or APOE ε4 (E4) into neonatal rodent brains.
24 % of human astrocytes showing hypertrophic morphologies and thicker processes surrounding Aβ deposits
12% showed atrophy
Proportion of human astrocytes undergoing morphological transformations and becoming hyper- or a-trophic was lower in the chimeric AD brains than in the brains of AD patients - allows us to study early stage of disease - not possible by tracking purely in-vivo
Study failed to implicate importance of APOE genes with other elements of human pathophysiology - perhaps animal model does not facilitate such human specific disease mechanisms
Interesting to perform RNA sequencing analyses at single-cell resolution to dissect the cellular states of transplanted astrocyte -> not possible due to limited recovery after transplantation.
Bao et al 2020 CoV2
The hACE2 transgenic mice poorly model the respiratory aspect of SARS-CoV-2 as shown in preliminary work with hACE2 mice - completely useless at modelling any other aspect of the disease. Studies from the Netherlands and France suggest that clots arise in 20–30% of critically ill COVID-19 patients, however this aspect of pathophysiology cannot be modelled in mice, who are generally resistant to thrombosis.
Matson et al 2018
Faecal microbiome modulates response to ICB in metastatic melanoma
Significant association was observed between commensal microbial composition and clinical response
Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy.
But germ free mice kept germ free through entirety of development (powerful antibiotics and sterile environments)
Significant reductions in bacterial load are associated with shifts in cell populations, signalling pathways, and organ morphology - likely develop cancers in very different ways
Livesey et al 2012
Human iPSCs manipulated to form stem cell cortical rosettes
Synaptogenesis occurs in vitro visualised with super-res microscopy and pre/post synaptic protein expression and tested functionality by observing patch-clamp spontaneous firing
Activity independent synaptogenesis - we know in human - activity dependent - generation of plasticity and appropriate behaviour may be challenge
Epilepsy
Organ-on-a-chip Zhou et al 2019
Using a Biowire chip seeded with cell-hydrogel mixture, have constructed a platform which can generate atrial- and ventricular-specific cardiac tissue by combining the directed cell differentiation and electric field conditioning, which is very close to the simulation of distinct electric signaling in adult heart chambers.
Kishoreet et al 2014
Used voltage clamping to assess relative interneuron contribution at different phenotypic swimming speeds
Discovered - hybrid model of motoneuron recruitment during locomotion - different pools are recruited at different speeds
Supported the focus on synaptic connectivity in understanding spinal cord circuitry
Alzubi et al 2017
HOWEVER sequencing found in humans and apes large overlap between these two TFs in sensory areas, while in mice this was not the case
Perhaps the expansion of cortical COUP-TFII expressing territory in human fetal brain mirrors the increased size and complexity of the association areas
Overlap with SP8 allows for association between sensory and motor areas, with them interconnecting.
Mice do not have gyrencephalic brain so poor models for studying complexity of brain
Primates more appropriate
Guerra et al 2008
Knocked-out Klrk1 in NK cells in mice, leading to NKG2D deficient NK cells.
This work was crucial in elucidating the role of NKG2D-dependent immunoediting in tumour surveillance in transgenic models representing spontaneous epithelial and lymphoid malignancies, and the role the immune system plays in suppressing cancer.
However, interpretation of the phenotype of knockout mouse is complicated by numerous factors, such as compensation for the knockout.
Any observed phenotype may have not arisen due to the mutation, but due to the genetic background of the mouse - genetic drift occurs upon breeding which eventually leads to the emergence of are phenotypically different substrains.
Mahajan et al 2016
They wrongly associated an immune deficiency phenotype to a knockout allele, when in fact it was a due to a mutation in the particular C57BL/6 substrain that the knock-out mouse was backcrossed onto.
The phenotypic difference in the substrain used was only discovered after a two-year investigation into why the laboratory could not reproduce previous results with a C57BL/6 substrain from a different vendor.
Scientists must be aware of this
Avian embryo FHF/SHF
Chicks grow inside eggs so can observe embryo with far less disruption than in-utero disruption ( in humans only possible with ultrasound that has extremely low resolution)
Identification of two heart fields (FHF/SHF)
Retrospective clonal analysis
Modified LacZ (LaacZ) expressed under primitive early marker (crescent)
duplication which renders the protein inactive, this can be spontaneously repaired to give a functional protein
Once repaired, all descendent have the repaired gene so that they will show up blue – derived from single event (clonally related) – size of colony reveals how long ago
Looking at many embryos - two distinct groups of clones of a particular size, either left or right ventricle, cells never overlapped (only overlap were huge clones which showed that they shared cells very long ago)
Drosophila SC model
Processes in cell biology very challenging to study (I.e trafficking and secretory systems) – too small to see without EM – therefore cannot be in-vivo study – lose dynamics of the system
Observing exosome biogenesis and trafficking
Drosophila male accessory gland secondary cell model overcomes these limitations as cells are large enough to be imaged with fluorescence light microscopy
Cells can be live imaged and organelle details can be visualised by expression of GFP under promoter
Fan et al 2020: Rab11-labelled multivesicular bodies make exosomes via an ESCRT-dependent mechanism in Drosophila secondary cells
Bates et al 2001
Demonstrated that in children aged between 5-8 with early unilateral brain injury, their difficulties in language (assessed using the mean length of utterance) seemed to resolve compared to age/gender matched controls.
Whereas adults with LHD don’t exhibit the same capacity for resolving these language deficits. This implies that children have a greater capability for plasticity which accounts for their ability to compensate largely in language deficits compared to adults. This work would have been impossible to carry out in animals
Human challenge trials
Edward Jenner success
In 2011, the Oxford University Centre for Clinical Vaccinology and Tropical Medicine ran HCTs exposing forty-one adults to pathogenic Typhi following vaccination, which was seen to be effective and is now recommended by the WHO for those living in regions of endemic typhoid.
Lead to approval of drugs far more rapidly than animal models
But unrepresentative population (young healthy adults) when in reality older people (65+) would be treated
Directly contradicting medicine’s central principle to “do no harm”. Jenner’s study would today be deemed unethical- he infect an 8-year old boy with a live untreatable virus, however eradicating smallpox is still considered one of humanity’s greatest achievements.
Today, human challenge trials are only considered when the disease can be objectively detected and effectively treated to prevent significant morbidity, such as in malaria.
Yamanaka
Reprogramming of human cells (normally fibroblasts) with administration of TFs (Oct4, Sox2, Klf4, and c-Myc) to induce pluripotency.
Understanding of development meant such cells could be generated
Was retroviral approach but recent concerns with mutations and oncogene activation - focus now on integration free methods.
Non integrating viral vectors including the Sendai virus.
Cytoplasmic RNA virus, will not deliver to nucleus, no chance of mutagenesis, cell recognises virus and produces IF response to remove it - by then the cells intrinsically maintain pluripotency.
iPSCs seizures
Drug-induced seizure is a major reason for compound attrition during the development of CNS drugs, usually identified during pre-clinical seizure-liability testing.
Currently, rodent ex vivo and organotypic hippocampal slices (which retain similar cytoarchitecture and connections as in vivo brain system) and primary cultures are the most commonly used in seizure-liability studies.
Controversy over the relevance and efficacy of these models, for example the lack of human receptors which may result in misleading data, has led to interest in the development of human-derived models.
Itzhaki et al 2011
Drug screening using LQTS human iPSC‐CMs.
Nifedipine: Ca2+ channel blocker - anti‐arrhythmogenic, normalised action potential duration - blocked spontaneous membrane depolarizations
voltage clamping
but no environmental influence, single cells not tissue
