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In vitro (Fach) / Zellbio (Lektion)

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• Comparison of CRISPR and RNAi screens 1. casTLE score: large positive: high-confidence increase in growth rate;                      large negative: ~decrease (essential) 2. in gene perturbation experiments: CRISPR more distinct phenotypes (DNA k.o.);                                                         RNAi incomplete knockdown, may retain gene function>obscured effects 3. access to non-coding regions 4. Cas9: gain of function
• Detection of RNA transcript in live cells & applications RBMB: red RNA transcript DAPI: blue nucleus rhodamine-labeled RNA: green targeted mRNA Applications: cell trafficking of individual RNA species; velocity measurement of transcript movement
• Ribo-Seq Translation measure of protein synthesis; reveal translational regulation & dynamics
• protein translocation by signal sequences
• protein turnover/degradation degradation: ubiquitin dependent turnover measurement: by measure the loss of radioactivity over time; affinity purification coupled with autoradiography; mass spectrometry (loss of 12C14N and accumulation of 13C15N)
• pulse chase parallel quantification of mRNA and protein turnover, pulsed labelled with:  protein: with SILAC mRNA: with 4sU then mass spectrometry and next-generation sequencing
• Dynamics of relationship between mRNA and protein During transition, delayed synthesis between mRNA and protein; For short-time adaption, delayed transcription and translation; At single-cell scale, transcriptional bursting, becomes visible, longer half-lives of proteins
• levels of gene expression and regulation genome > transcription > RNA processing and nuclear export (all in nucleus) > translation > posttranslation (all in cytoplasm)
• gene amplification and deletion before gene become available for expression: rara event amplification: Xenopus laevis, selective ampli of nucleoli of genes encoding rRNA deletion: erythrocytes, ennucleated after sufficient synthesis of hemoglobin mRNA;               copepoda, heterochromatic region excised (except gametes)
• decondensed chromatin genome level: sensitive to DNase I digestion
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• spatial chromatin organization and transcriptional activity karyotyping gene-rich regions concentrate towards the interior of nucleus gene-poor region: near nuclear membrane (nuclear lamina); nuclear lamina-mediated gene silencing, low transcriptional activity radial positioning of genes nuclear pore complexes: site of transcription
• cis-reporter system cis-acting response element + TATA box + reporter extracellular signal activate protein kinase (PK);  PK phosphorylate(=activate) trans-activators (TA); PO4-TA bind response element; reporter gene expressionn
• reporter gene luciferase & beta-gal transfection method: nucleofection (kinetics: luci max. 6-16h post transfection; gal sustain for days)                                   lipids (luci max. at 24h post)
• multiplexed dual reporter assay wells 45 verified signaling pathways measure downstream TF activities dual luciferase, positive and negetive control Applications: pharma, cancer, immunology
• full-length reporter-tagged mammalian genes multiple clustered enhancers + (interspaced with) silencer, insulator + core promoter (TATA, INR, DPE) (INR: initiator sequence; DPE: downstream promoter element) features: tagged proteins express at physiologically correct times and levels; physiologically appropriate splicing isoforms are produced; tagged proteins exhibit native subcelluar localization and native biological function.
• gene tagging by CRISPR for image-base screens: GFP, endogenous reporter; monitor changes in localization or stability: endogenously tagged; combi of reporter and tag.
• ChIP-chip/seq chromatin immunoprecipitation for identify regions of TF binding/chromatin-protein interaction cells(DNA) fixed with formaldehyde to crosslink DNA to protein(of interest) > DNA fragmentation (DNA-protein complexes) > immunoprecipitate with antibody against protein > reverse crosslinking, DNA amplification, hybridization
• ChIP-seq applications single locus analysis (nanog) oct4, sox2 and nanog enhancers motifs
• deep seq tech examples: TF binding sites: ChIP-seq alternative splice variants: RNA-seq open or accessible regions in euchromatin: DNAse-seq
• microRNA in translation: translational repressor
• cDNA microarrays (transcriptome) control cell + cy3 (green) + exprimental cell + cy5 (red) mRNA extracted and reverse transcription
• qRT-PCR array for pathway and disease analyses human peripheral blood mononuclear cells; intracellular cytokine production
• nanopore DNA, RNA, protein real-time data; rapid; direct analysis of molecule of interest; multiplexing and automation
• miRNA, antisense oligonucleotides miRNA bind target mRNA, leading to degradation of mRNA or translation inhibition, partially complementary to target; antisense oligonucleotides: bind mRNA, RNase H degrade DNA/RNA heteroduplexes
• siRNA off-target effects microRNA-like off-target silencing is sequence-specific; results from imperfect pairing of siRNA strands with sequence motifs that reside primarily in 3ÚTR regions of cellular mRNAs reduce OTE by siRNA pooling
• seed effect RNAi screen phenotypes in RNAi screens may largely be a consequence of seed-based off-target effects
• arrayed vs pooled screening formats differ in the way in which the targeting reagents are constructed and how cell targeting and readout is carried out. arrayed: reagents are seperately synthesized and constructs are arranged and cell targeting in multiwell plates; viral transduction; readout (cell count) measurements individual well  pooled: synthe and constructed as pool; viral integration; readout (cellular signature) through NGS, based on comparison of abundance
• imaging tech epifluorescence microscopy confocal ˜ TIRF (total internal reflection fluro)˜
• B cell receptor (BCR) complex FRET: interactions of BCR chains cuz the BCR signaling is not well established; all possible combi from BCR's chains (lg, lga, lgb) engineered to contain fret donor CFP and fret acceptor YFP.
• cell substitutes for cell-cell interaction studies substitutes for lymohocytes activation etc. (change Antigen-presenting cell (APC) ) coated beads (antibody); coated coverslip (coat with antibody); supported lipid bilayer (peptid-MHC complex)
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• B cell antigen binding visualization supported lipid bilayer with antigen B cell form immune synapses with the antigen TIRFM imaging & FRET (microcluster association)
• BiFC BCR signaling etc. monitor the dimerization of tagged proteins by means of assembly a complete YFP domain from the amino-terminal and C-terminal half domains of YFP and CFP
• FRET, confocal microscopy, TIRF applications in live cell imaing:  FRET: conformational changes confocal: intracellular localization cell adherence to surface: TIRF
• finite cell line limited lifespan & cell division times, retains diploid karyotype of donor tissue
• cell cycle progression cdk activation; cyclin; G1>S>G2>M
• senescence and characteristics via activation of p53 and pRB tumor suppressor pathway stress response replicative-, oncogen-induced-,stree-induced senescence irreversible growth arrest: SASP, DNA-SCARS, DDR, SAHF(DAPI red, HP1-gamma green), SA-Bgal  (SA: senescense-associated; SCARS: segments with chromatin alterations reinforcing senes; DDR: DNA damage response; SAHF: ..heterochromatin foci; Bgal: bata-galactosidase) short term: tumor suppression and tissue repair long term: tumor promotion and aging
• pRB signaling pathway activator of senescence (by shutting down transcription of E2F target genes) Rb controls expression of proteins to pass late G1 check point and initiation of S phase retinoblastoma
• permanent cell line = continuous cell line not immortilized line transformed spontaneously; indefinitely divide; aneuploid chromosome number; heteroploid cell population; reduced cell cycle duration; less complex media (serum free); loss of density limitation (multilayer growth); loss of differentiation
• transformation of permanent cell lines spontaneous or induced permanent phenotypic change due to heritable DNA alterations characteristics: genetic instability; immortality; abberant growth; malignancy
• filter well invasion assay boyden chamber: upper and lower compartment, ECM/collagen membrane calcein fluorescence
• HMF HMEC mamary fibroblast: finite; phase a,b epithelial cells: permanent; phase a,b,c,d a: logarithmic growth b: growth plateau c: second epithelial proliferation d: seconf epithelial growth plateau > cells after phase d have undetectable expression of p16 and have chromosomal instability
• transition from finite to permanent spontaneous: rare in human, frequent in rodents active immortalisation by: (pre)carcinogenic chemicals (benzoapyren);                                        recombinant telomerase (retain original phenotypic traits to some extent);                                        viral oncogenes (large T antigen from SV40)
• human papilloma virus HPV E6 & E7 together immortalize early cultures (preselection phase, less passaged cells) as well. E6 binds to p53 and promote its destrction; E7 binds to Rb and disrupts its ability to halt cells at the restriction point (G1>S)
• telomeres, M1, M2 (cellular state) M1: replicative senescence; hayflick limit; abnormal growth, tumorigenesis (p53 inactivation etc.) M2: crisis; telomeres critically shortened, genomic instability, cell death after M2: immortal cancer, telomeres stabilized
• SV 40 large T, hTERT human meibomian gland epithelial cells immortalized with both was abnormal
• ambivalent function of Myc in DDR Myc enhances DDR signaling and stimulate DNA repair (inducers as well as suppressors of senescence)
• cross contamination contamination with different cell lines or different levels of the same line analysis: morphology; growth rate; short tandem repeat markers; DNA fingerprint solution: cloning of identified target cells and subsequent verification
• HGSOC models high grade serous ovarian cancer models average properties and selected genetic events can be used to distinguish better and poorer models average properties: histological subtype; citation frequency in literature; altered fraction of genome; number of mutations per milion bases.. selected genetic events: alterations recurrently found either in HGSOC; one of the three other major subtypes of ovarian cancer
• stem cell niches niches: different cellular components, secreted factors, immunological control, ECM, physical parameters, metabolic control Interactions between stem cells and their niches are bidirectional and reciprocal
• experimental considerations substrate functionalization 3D culture models population vs single cell analysis

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