Transcript tools in develoomental biology

Applied Developmental Biology
Dr. Lubna Tahtamouni
The Hashemite University
2010
Week # 2
Tools in Developmental
Biology
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Questions to be answered in Dev. Bio.:
- axis determination (AP, DV, LR)
*Q. how do you ‘break’ the symmetry of the egg? Sperm
entry, localized determinants
- cell differentiation, cell proliferation, cell growth
- cell migration, polarization (symmetric vs asymmetric cell
division), cell shape (giving cells different morphologies, e.g.
neurons), cell death
- morphogenesis (how cells come together to form tissues;
and how these tissues migrate)
- organogenesis
- timing (biological clock)
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Questions to be answered in Dev. Bio.:
-aging
- germ cell development, fertilization
- stem cells (what are they (multipotency, ability to selfrenew), why did they become so ‘trendy’ (Dolly the sheep,
1997, showed that cloning was possible; development of
human ES cells, 1998)
- regeneration
- How efficient is human development? (talk about
implantation)
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Developmental biology Model systems
Worm (C. elegans)
Fly (Drosophila melanogaster)
Fish (zebrafish, medaka)
Mouse
Xenopus (laevis, tropicalis)
Chick (Quail)
Sea urchin (echinoderm)
Many systems available: which one to use: most powerful one where you can
study your questions of interest. (also, many of these model systems are used
to study processes other than developmental biology).
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Drosophila melanogaster
Arbacia punculata
Gallus domesticus
MODEL SYSTEMS
for the experimental
analysis of development
Mus musculis
Xenopus laevis
and tropicalis
Caenorhabditis elegans
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One of the answers to some of these questions:
Differential Gene Expression
- DNA is the same in all cells: genomic equivalence
- Some genes are mutated, or silenced but not lost!!!!!!!
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Central Dogma of Life
Reverse Transcription
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How to prove that some genes are expressed and some are not?
-Test for TEMPORAL/SPATIAL expression of RNA
•Blotting
•In situ hybridization
•PCR
- Test for the function of a certain gene
•Gain of function: microinjection
Transfection
Electroporation
Transgenic animals
• Loss of function
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Staining to find
-how the cells look
(anatomy)
TISSUE
CELLS
Histology
Cell
Biology
Proteins
Biochemistry
Western Blotting
-To detect proteins
DNA
ELISA
-To quantify
proteins
Enzyme Assays
-To measure
enzyme activity
Southern Blotting
-To find copy number
of genes
RNA
Northern Blotting
Real-Time PCR
-To study gene
expression
Genome Sequencing
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Blotting Techniques
•
Northern Blotting ( mRNA expression)
•
Southern Blotting (copy number)
•
Western Blotting ( protein expression)
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Western Blotting
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Western Blotting
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E
Enzyme
L
Linked
I
S
Immuno
Sorbent
A
Assay
Protein molecule that performs a
chemical reaction
Linking an enzyme to an
assay/test
Attachment of antibodies
Test to find out something
 Technique based on antigen-antibody reaction
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Well with antibodies
Well with antibodies and
BSA added
antigen binding sites
Well with antibodies,
BSA, and test sample
Well in a microtiter plate
Antibody structure
Well after washes with
wash buffer
Well after adding substrate
Color developed due to the
formation of a substrate
Secondary antibody linked to
an enzynme is added to the well
Well after removing
excess antibody
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Southern / Northern Blotting
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In situ hybridization
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PCR
RT-PCR
Real-Time PCR
-To study gene
expression
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PCR : Polymerase Chain Reaction
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Reverse TranscriptasePolymerase Chain Reaction
End of 35 cycles
236 = millions of
copies
PCR products
RT
RNA
Marker
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4 copies
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8 copies
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16 copies 32 copies
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Preparation of cDNA or first strand RT
Reverse Transcription
5’ GACCCAAUUGGUCAGCUAAAAAAA 3’
mRNA
5’ GACCCAAUUGGUCAGCUAAAAAAA 3’
Reverse
transcriptase
……TTTTTTT 5’
A, T, G, C
dNTPs
Reverse
Transcription
3’ CTGGGTTAACCAGTCGATTTTTTT 5’
1ST strand cDNA (complementary DNA)
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Fate mapping
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Gain-of-Function
Transgenic animal
Loss-of-Function
RNAi
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Gain-of-Function/Trangenics
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Loss-of-Function/RNAi
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Loss-of-Function/RNAi
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THE END
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