gene function analyses, reporter genes in direct and reverse genetics
DESCRIPTION
Gene function analyses, Reporter genes in direct and reverse genetics. Reporter genes. encode proteins that can be directly visualized or enzymes, whose activity can be visualized quantitative or qualitative assessment - PowerPoint PPT PresentationTRANSCRIPT
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(!)