gene function analyses, reporter genes in direct and reverse genetics
Post on 02-Jan-2016
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Reporter genes
• encode proteins that can be directly visualized or enzymes, whose activity can be visualized
• quantitative or qualitative assessment• promoter activity analyses, subcellular localization of
proteins, optimizing transformation procedure, ….
Constrains:- background (autofluorescence, natural enzyme activity
within the tissue) - protein stability (mask changes in promoter activity)- degradation of fusion proteins
Reporter genes
gene product substrate detection
gusA -glucuronidase (E. coli) MUG fluorescence
X-gluc histochemical
luc luciferase (fire fly) luciferin luminiscence
gfp green fluorescent protein xxx fluorescence
(gelly fish)
Fluorescent proteins: GFP, DsRed, mCherry, EosFP
Aequoria victoria
Barevné varianty GFPa DsRed
- unique tools for in vivo labelling
- encoded with small genes
- origin: sea Coelenterata (corals, gelly fish)- modified forms:
- fluorescent features, - codon usage, splicing, stability, …
EosFP – photoactivatable fluorescent protein (green to red FP)
GUSQualitative detection (X-gluc)• oxidized blue precipitate of reaction product• low background• slow diffusion• mostly in fixed material
(X-gluc = 5-bromo-4-chloro-3-indolyl glucuronide)
Quantitative detection (MUG)• GUS enzyme isolation, fluorimetric statement• highly sensitive, low background
(MUG = 4-methylumbelliferyl-beta-D-glucuronide)
Gene function analyses
Modulation of expression:- increased protein level (overexpression) –
introduction of a gene with a strong constitutive promoter
- alt. gain-of-function mutations
- decreased protein level by RNAi
- alt. loss-off-function mutation
Tilling – point mutation in commercial collections
Reporter gene fusions
Decreasing protein levelInduction of RNA interference (dsRNA formation): 1) antisense RNA2) hairpin RNA (e.g. sense-intron-antisense) 3) non-terminated RNA (dsRNA via RdRP)
- dsRNA cleavage by DCL, siRNA formation, sequence specific mRNA degradation or block of transcription due to promoter methylation
+
RdRP
Intermolecular pairing
intramolecularpairing
complementary strandsynthesis
Promoter analysisFusion of analyzed promoter with reporter gene (transcription fusion), with or without the original transcript:
Indicates: - tissue, organ, developmental specificity- responses to external factors
Confirmation with other approaches advisible (risk of artifacts)
P gen T
reportérový gen
- usually introduction of new copy into the genome
Fusion protein formation
- stop codon removal, fusion in reading frame (= translational fusion)- functional domains, natural interaction(!)
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