Lecture 12 - Drosophila segmentation (Debrief session) Flashcards
(28 cards)
The lacZ reporter gene is carried on a ‘transgene’. What is a transgene?
Transgene is a section (or sections) of genetic material transferred from one organism into the genome of another.
How is transgenesis carried out in Drosophila?
(i) transposons e.g. P-elements
(ii) site specific recombination e.g. attB/attP
(iii) homology-dependent recombination - e.g. at sites of double-strand DNA breaks produced by CRISPR/Cas9
How does Germline transformation occur?
DNA is injected into the posterior of pre-cellularised (fertilized) embryo - in region of pole cells, before they start forming.
How does transgenesis occur via a P-element?
‘copy-paste’ mechanism
- integration into random genomic site
What is phiC31-mediated site-specific recombination?
Integration into previously attP acceptor site
How does homology-dependent recombination (HDR) at CRISPR/Cas9-induced double-strand breaks (DSB) function?
HDR insertion site determined by:
- gRNA sequences specifying DSB site
- homology arm sequences
- utilize DNA template
Particularly useful for genome modification - e.g. insertion of GFP
How can transgenesis occur in a zebrafish?
(i) transposons e.g. Tol2
(ii) site specific recombination - attP/attB
(iii) homology-dependent combination e.g. at sites of double-strand DNA breaks produced by CRISPR/Cas9
(iv) injection of DNA & random integration (at a low efficiency)
How can transgenesis occur in a mouse?
(i) injection of DNA & random integration
(ii) homology-dependent recombination in mouse embryonic stem (ES) cells & injection into embryonic blastocysts
(iii) but also sometimes specific recombination & now HDR at double-strand breaks via direct pro-nuclear injection
How does traditional PI-based transgenesis function?
Linear DNA fragment
Transgene expression is variegated. This method requires that multiple mouse lines be analyzed.
(3 months)
How does ES cell-based targeted transgenesis function?
Targeting vector
Transgene expression is reproducible, analysis of a single germline transmitted mouse line is sufficient.
(6-8 months)
How does PI-based targeted transgenesis function?
Donor vector + recombinase
Transgene expression is highly reproducible & faithful, analysis of a single transgenic mouse line is sufficient
Why does the tissue need to be fixated with a protein cross-linking agent, such as formaldehyde?
Cross-linking joins together nearby proteins, creating a stable structure that can withstand the subsequent washes
What does the detergent in the wash buffer (PBT) do, and why is it necessary to do this?
Detergent breaks down the cell membranes (primarily lipids) & allows penetration of probes into the embryo.
What would happen if we didn’t fix the tissue before exposing it to wash buffer?
Without fixation, detergent would wash away many of the protein components of the cells, causing loss of tissue structure (and also loss of e.g. B-galactosidase enzyme)
How does fixation with formaldehyde occur?
- Formaldehyde is smallest aldehyde
- acts primarily on the amino groups of amino acids (e.g. lysine) & nucleic acids, and the nitrogen atoms of peptide linkages
Why are the embryos mounted in 30% glycerol, instead of in aqueous solution?
- Glycerol ‘clears’ tissue, rendering it more transparent
- Clearing occurs because 30% glycerol has a higher refractive index than water, resulting in smaller differences in refractive index between the solvent & the tissues of the embryo, which in turn results in less light scattering.
Glycerol is also viscous, making the preparation a bit more robust
In this practical we used a lacZ reporter gene to reveal the pattern of transcript expression of a gene
lacZ = bacterial gene encoding B-galactosidase enzyme
The B-galactosidase substrate X-Gal is cleaved to yield galactose, which forms an insoluble blue precipitate
How else can the regional expression patterns of gene transcripts within an embryo be revealed?
- RNA in situ hybridisation
- complementary DNA or RNA probe hybridized to the tissue
- probe labelled to allow detection e.g. by an enzymatic or fluorescent method
How does probe detection function?
- target mRNA
- DIG-labeled probe
- anti-DIG-AlkPhos
- reaction product
Digoxigenin (DIG) epitope with enzyme-conjugated anti-DIG
- alkaline phosphatase (AP)
- organic fluorophores
Use 2 probs with different labels to detect 2 transcripts
How does fluorescent immunolabelling?
- target protein
- primary antibody
- secondary antibody
- fluorochrome
Describe the segmentation gene hierarchy that acts during Drosophila segmentation. What are the functions of the Gap genes, the Pair-rule genes & the segment polarity genes
Maternal Genes
- bcd & nos mRNAs anchored at A & P poles
- Bcd & Nos proteins form gradients in embryos
Gap Genes
- hb, Kr, Kni, Gt genes activated at different Bcd concs
- overlapping Hb, Kr, Kni, Gt domains divide embryo
Pair-rule Genes
- maternal & gap protein code activates pair-rules
- 7 stripes with different enhancers
Segment Polarity Genes
- pair-rule genes turn on segment polarity genes
- expressed in 1 cell of each segment
What occurs in a wingless mutant?
cells making “naked” cuticle are mis-patterned are lost (death) or redirected to form denticles
Loss of Wg causes a denticle ‘lawn’ phenotype, but also some cell death
What occurs in Gap gene mutants?
- Kruppel
- hunchback
- knirps
Lack different regions
Loss of Hb results in loss of T2 & T3 segments, so abdominal segments are shifted anteriorly
When do we see staining of genes?
30-60 minute delay after transcription until see X-Gal pattern
hunchback & fushi tarazu appear in 11/12th mitotic cycles during stage 4, but do not appear till an hour later in staining.
