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Vertebrate development > Techniques 1 > Flashcards

Techniques 1 Flashcards

1
Q

Classical methods

A

Morphological descriptions
Histology
Cell ablation
Study of mutants

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2
Q

Morphological descriptions

A

Isolate embryos at different stages and describe their morphology

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3
Q

Histology

A
  1. isolate embryo
  2. fix using fixative e.g. paraformaldehyde
  3. dehydrate
  4. embed in wax
  5. section + take onto slides
  6. stain + observe tissue histology under microscope
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4
Q

Fixation

A

Kills tissue to prevent postmortem decay

e. g. using paraformaldehyde
- > cross-links proteins

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5
Q

Cell ablation

A

Ablation of specific cellular structures in embryo + allow it to develop
- study regeneration, development + compensation

Use lasers or toxins to kill cells

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6
Q

Study of mutants

e.g. Taplid chick

A

Taplid3 = chicken mutant w/ abnormal limb patterning + malformations in regions of embryo known to depend on Hedgehog signalling

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7
Q

Study of mutants

e.g. Mouse piebald mutation

A

Broad white patches on belly + head

Pigment called melanocytes in mouse embryos have mutations in c-kit gene

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8
Q

Lineage tracing

A

Identify all progeny of a single cell

Essential for studying stem cell properties

Help to understand tissue development, homeostasis + disease

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9
Q

Tissue homeostasis

A

Balance between cell division + cell differentiation

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10
Q

Fate mapping + lineage tracing of embryos

A

> Grafting
Vital dye marking
Fluorescent dye + radioactive labelling
Genetic marking + grafting

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11
Q

Tissue grafting

e.g. Spemann + Mangold

A

Graft a tissue from 1 embryo to another

-> graft of organiser led to conjoined twin embryos

.:. embryo’s cells are not committed to a certain fate

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12
Q

Naturally occurring genetic markers as lineage tracers

e.g. quail and chick cells

A

Cells from quail embryo grafted into chick embryo

- can determine the cell type from their heterochromatin of their nuclei
quail cells have a large nucleolus

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13
Q

Methods for identifying genes regulating development

A

> Homology searching
Cloning of cDNA and screening of libraries
Screening + sequencing of expressed sequence tags
Mapping + identifying genes responsible for a mutant phenotype

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14
Q

Methods to detect gene expression

A
  1. mRNA
    - northern blotting
    - RT-PCR
    - microarrays
    - in istu hybridisation
  2. Protein
    - Immunostaining
    - Western blotting
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15
Q

Molecular hybridisation

A 
Southern blot (DNA-DNA)
Northern blot (DNA-RNA)
Western blot (RNA-RNA)
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16
Q

Southern blot

A
  1. Restricted DNA on gel
  2. Denature + transfer to filter
  3. Hybridise with probe
    = labelled DNA
17
Q

Northern blot

A 
1. RNA on gel 
(separated by gel electrophoresis)
2. Transfer to filter
3. Hybridise with probe 
= labelled DNA
18
Q

Western blot

A
  1. Protein on gel
  2. Transfer to filter
  3. React with probe
    = antibody

Shows which proteins are expressed

19
Q

Analysis of gene expression

A 
> Northern blotting
> PCR
> In situ hybridisation 
a) sections 
b) whole mounts
> microarray analysis
20
Q

Expression of RNA

A 
> Quantitative assays
- in situ hybridisation 
> Qualitative + tissue specific assays
> Large scale assays 
- micro assays
21
Q

RT-PCR

= reverse transcriptase - polymerase chain reaction

A
  1. cDNA synthesised by reverse transcriptase of mRNA
  2. RNase removes mRNA
  3. PCR - using primers specifically binding to sequence of interest
  4. visualisation on agarose gel (after staining DNA)
22
Q

Microarray

A
  1. isolate mRNA from samples
  2. RT + fluorescent labelling (diff colours for diff samples)
    - > cDNA
  3. Hybridisation to microarray
4. Chip contains sequences representing diff genes
On chip:
Green = mRNA only present in sample 1
Red= mRNA only in s2
Orange = mRNA in both 
Black = mRNA in neither
23
Q

In situ hybridisation

A

This method either:
1. localises mRNA within cytoplasm
or 2. DNA within chromosomes of nucleus

Hybridise sequence of interest to a complementary strand of a nucleotide probe

  1. Labelled DNA or RNA probe (e.g. with digoxigenin) complementary to target mRNA
  2. Labelled antibody (e.g. anti-digoxigenin) binds to cDNA-probe complex
24
Q

Methods to manipulate gene expression

A

> Overexpression/ ectopic expression of genes:
- transgene expression systems

> Reduced gene expression

  • dominant negative proteins
  • gene targeting by homologous recombination
  • RNAi
  • morpholinos
  • mutagenesis screens
25
Q

Methods to detect + manipulate gene expression

A

Gene trap/ enhancer trap

26
Q

Levels gene expression can be detected at…

A

mRNA

Protein
- needs antibodies that specifically bind to protein of interest

27
Q

Identifying function of a gene…

A

Manipulate gene expression + observe effects

28
Q

Protein expression methods

A 
> isolation, separation + purification of proteins
> antibody production 
> Western blotting
> proteomics
> immunohistochemistry
29
Q

Localisation of mRNA + protein can be different from each other
e.g. Sonic hedgehog in neural tube

A

SH mRNA is made in cells of notochord + floor plate
-> cells detected by in situ hybridisation using probe

SH protein secreted + forms ventral to dorsal conc grad in neural tube
-> protein gradient detected by immunostaining using antibody

30
Q

Western blotting antibodies

A

Commonly labelled with HRP (horse radish peroxidase)

- catalyses a chemical reaction associated with light emission

31
Q

Proteomics

A
  1. Preparation of protein extracts
  2. Isoelectric focusing
  3. Polyacrylamide electrophoresis
  4. MALDI-Tof
  5. Data mining
32
Q

Techniques to analyse gene function

A
  1. Introduce cloned genes into cells by transfection
  2. Generate transgenics by intro of DNA into fertilised eggs
  3. Generate mutants by homologous recombination in ES cells
  4. Regulate gene expression by transgenic analysis
  5. Knockout gene function by antisense RNA
  6. RNA interference

Vertebrate development (12 decks)

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