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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Gene function analyses, Reporter genes in direct and reverse genetics

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Gene function analyses reporter genes in direct and reverse genetics

Gene function analyses,Reporter genes in direct and reverse genetics


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 substratedetection

gusA b-glucuronidase (E. coli) MUGfluorescence

X-gluc histochemical

luc luciferase (fire fly)luciferinluminiscence

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)


Gene function analyses reporter genes in direct and reverse genetics

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


Gene function analyses reporter genes in direct and reverse genetics

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


Gene function analyses reporter genes in direct and reverse genetics

Promoter fusion with GFP and GUS

Arabidopsis

thaliana


Gene function analyses reporter genes in direct and reverse genetics

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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