Genetic Mosaic/Chimeric Analysis in the Zebrafish. Mosaic: An organism consisting of cells of more than one genotype (derived from the same individual). Chimera: An organism consisting of cells derived from more than one individual. What is chimeric analysis used for?
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An organism consisting of cells of more than one genotype (derived from the same individual)
An organism consisting of cells derived from more than one individual
A gene is required in the cells that exhibit the mutant phenotype
Gbx mutant has no cerebellum
Gbx mutant cells are excluded from the cerebellum
A gene is required in cells other than those that exhibit the mutant
Phenotype: motor axon guidance
Jing et al., Neuron 2009
In the fly: genetic tricks: flp-mediated mitotic recombination creating homozygous mutant clones.
In the mouse:
aggregation of blastomeres from pre-implantation embryos
Cre-mediated tissue-specific recombination (“tissue-autonomy”)
In the zebrafish:
Transplantation of cells between wild-type and mutant (or morphant) embryos.
Cell-type specific expression of a dominant-negative protein
Cell-type specific rescue of a mutant phenotype
Woo and Fraser, 1996
- it’s the ‘perfect’ experiment for a grant proposal- each outcome is informative
- often reviewers will ask for it, so why not do it right away
- despite that this is a powerful approach, only few papers include such data
Purpose of the lab: to show you that this is a powerful technology, that
does not require much…..
What will we do today:
- place & orient donors and hosts into single well agar molds
H2A-GfP (ubiquitous, nuclear GFP); RhD
Isl1-GfP (motoneurons, cytoplasmatic GFP); RhD
mnx1-GfP (motoneurons, cytoplasmatic GFP) ;RhD
Brn3:GFP (RGC, cytoplasmatic GFP;
Ath5:GFP (RGC, cytoplasmatic GFP;
postepiboly embryos into 60 mm dish & keep at 28.5
Tips during transplanting:
-orient all embryos before placing the transpl. needle into the holder
- before taking up the first embryos, make sure the meniscus is steady
If not tighten all connections- the system has to be airtight in order to work
- keep meniscus low and in eyesight!
- do not ‘suck’ in donor cells with to much pull, slowly!
- don’t transplant >40 cells along margin.
- analysis of cell transplants: tomorrow 9am-11:30 pm!
• required for induction of endoderm and patterning of mesoderm
• rescued by injection of wt transcript (unusual case)
Germ line replacement chimeras
• A tool for identifying maternal functions for essential genes
- mRNA and protein accumulate during oogenesis
- zygotic transcription begins only hours prior to gastrulation
• also useful for studying double or triple mutant phenotypes, eg RNA-Seq
Example: One-eyed pinhead (oep)
• GOAL: propagate mutant germ line through WT host using blastula transplantation
GFP-nos1-3’UTR marks PGCs with GFP (unfortunately not yet at blastula stg.)
Deadend morpholinos eliminate the host germline cell autonomously
Cells don’t have to be transplanted to the margin
Cells have to be removed from the margin
Ciruna et al. PNAS 2002
AM: inject lineage marker into donor embryos
1:00 PM: demo: Michael Granato: blastula transplants on the dissecting microscope;
1:30PM Cecilia Moens: germ cell transplants
2:00PM- 4:30: Your turn.
4:30PM-6:00: Gastrula Stage Transplants with Cecilia
Tonight after the talks: mount selected chimeras for confocal time-lapse overnight.
(avoid too much caffeine)
Agarose molds for injection and transplants:
Adaptive Science Tools
31 Gifford DriveWorcester, MA 01606-3535
TU-1: six ramps containing one 90-degree and one 45-degree beveled side for micro-injection
PT-1: transplant mold with 150 divets to accept single embryos
World Precision Instruments MPH3 (specify outer diameter of pipettes and diameter of optional brass handle)
Fine Science Tools MM-33 (needs magnetic stand)
Or World Precision Instruments M3301R
Borosilicate Glass pipettes:
World Precision Instruments (match outer diameter to pipette holder)