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Differential Gene Expression in the Gastrula of Xenopus Laevis

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Differential Gene Expression in the Gastrula of Xenopus Laevis Powered By Docstoc
					Differential Gene Expression in
the Gastrula of Xenopus Laevis
   Differential Gastrula
    mRna – DG mRNA
   Transcribed during
    Gastrula stage;
    Selectively used
   Maternal mRNA –
    Passed from female
    parent to the egg.
Background: Early Development
   Egg > Embryo >
    Blastula > Gastrula >
    Neurula > Tadpole
   Gastrula - First
    appearance of three
    germ layers:
    Endoderm,
    Mesoderm, &
    Ectoderm
Experimental Goal:
   Determine and Isolate the genes
    responsible for early differentiation
   Separate, study DG mRNA
Method & Obstacle:
   Plan Of Action:      Examples:
    Hybridize mRNA to     If Blastula is tested,
    cDNA library           Maternal RNA has
    (Maternal & DG         strong signal
    mRNA) via Colony      If Gastrula is tested
    Hybridization.         DG RNA has strong
   Problem: 0.05% of      signal
    10000 mRNA too        Rare mRNA not
    rare for detection     detected
Solution & Methodology:
   Use of modified
    cDNA cloning
    procedure
   Use highly enriched
    DG cDNA Library
   Purify sequences by
    hybridizing to ovary
    mRna
Methodology Cont…
   Enriched DG cDNA
    inserted to ClaI site
    of pBR322 plasmid
    vector.
   Results in 150,000
    clones in pBR322
    vector.
Dot Blot Hybridization:
   Six clones were
    picked from
    “reference cDNA
    library” (+) control)
   pBR322 fragment (-)
   Hybridized to labeled
    probes from Egg,
    Blastula, Gastrula &
    Tadpole stage RNA
    Fig. 2
Southern Blot:
   DNA from 9
    nonhomologous
    clones labeled by
    nick translation
   Hybridized by
    Southern Blot using
    Eco-RI digest of
    Xenopus genomic
    DNA (Fig. 3)(in kb)
Northern Blot
   DG Clones and r5
    (probes) hybridized
    to Gastrula RNA
   Lane 42 proof of
    possible nuclear
    precursor molecules
   (in kilobases)
Nuclease Protection Assays
   DG mRNA is                 Unhybridized mRNA
    hybridized to labeled       degraded by
    ss DG 42 DNA                nucleases
    excess probes              Hybridized mRNA
                                visualized via
                                Autoradiogram
NPA continued…
   Measurements were
    compared to a
    control and DG clone
    concentration was
    calculated.
   Calculated
    concentration
    adjusted to dot blot
    data in Fig. 5
DG Abundance Table:
   DG Gastrula Calculated to be 48
    picograms per Gastrula
   Supports figure 2 data that DG mRNA is
    synthesized de novo.
Conclusions:
   Enrichment Cloning technique was a
    success
   Confirmed the presence of Differential
    Gastrula mRNA separate from Maternal
    mRNA
   Gradually disappear after Gastrula;
    Implication that it has little preceding
    stages. Some increase in concentration.

				
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posted:7/22/2012
language:English
pages:13