gene function analyses reporter genes in direct and reverse genetics
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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

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reporter genes
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 genes1
Reporter genes

gene product substrate detection

gusA b-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
Fluorescent proteins: GFP, DsRed, mCherry, EosFP
  • - unique tools for in vivo labelling
  • - encoded with small genes
  • origin: sea Coelenterata (corals, gelly fish)
  • modified forms:
  • - fluorescent features,
  • - codon usage, splicing, stability, …

Aequoria victoria

Barevné

varianty GFP

a DsRed

EosFP – photoactivatable fluorescent protein (green to red FP)

slide5
GUS

Qualitative 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
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

slide7
Decreasing protein level

Induction of RNA interference (dsRNA formation):

1) antisense RNA

2) hairpin RNA (e.g. sense-intron-antisense)

3) non-terminated RNA (dsRNA via RdRP)

Intermolecular pairing

+

intramolecular

pairing

RdRP

complementary strand

synthesis

- dsRNA cleavage by DCL, siRNA formation, sequence specific mRNA degradation or block of transcription due to promoter methylation

promoter analysis
Promoter analysis
  • Fusion 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)

reportérový gen

PgenT

- usually introduction of new copy into the genome

slide10
Fusion protein formation

- stop codon removal, fusion in reading frame

(= translational fusion)

- functional domains, natural interaction(!)

gfp fusion proteins
GFP fusion proteins

- Protein localization analyses, protein interactions

- in vivo labelling of cellular structures

Golgy complex chromosomes microtubules

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