Zebrafish Flashcards
Embryogenesis
Eggs are optically transparent, allowing easy visualization
Easy with microinjection -> you can see specific structures/cells
High rate of development in embryos (body axis visible after 24h)
The key developmental processes are similar across vertebrates(ryggradsdjur)
Heart regeneration:
Regeneration of cardiomyocytes in zebrafish is not based on stem cells or trans differentiation of other cells but on the proliferation of pre-existing cardiomyocytes. Compared to humans, in which cardiac injuries result in irreversible loss of myocardial cells and formation of fibrotic scar tissue. -> undermines pump function -> congestive heart failure. (Studies could identify specific molecules able to regulate proliferation in existing cardiomyocytes in humans)
Sex determination
In mammals, the sex of the progeny is determined by sex chromosomes, specifically the SRY gene in the Y-chromosome. In zebrafish there are several other contributions:
Diversity of loci (fixed position on chromosomes where particular genes or genetic markers are located)
All gonads go through a transient female-phase during early larval life. Sexual differentiation is initiated between 20 and 25 days post-fert. Females: oocytes continue to mature. Males: oocytes undergo apoptosis.
The Cyp19a1a gene encode Aromatase, which is crucial(indispensable) for oestrogen production. In males, this gene is inhibited. There are several genes involved in the male determination!
What is the advantage of Zebrafish?
Small and robust
Maintenance cost is lower than those for mammalian models
Hundreds of eggs at weekly interval
Rapid hatching of eggs
Optically translucent embryos
The egg are fertilized and develop outside the mothers body and is an ideal model for studying the early development
Easy for molecular and genetic analysis.
Zebrafish have the same major organs and tissues as humans.
Disadvantages
Just a fish
Takes long time to sexually mature
Several mammalian organs are missing
Cold-blooded; not physiologically identical to humans
Greatly influenced by their environment; temperature, population density, water quality, nutrition, lightning
Forward genetics/Random mutagenesis
Most genetic screens are performed with ENU(N-ethyl-N-nitrosourea) which induces genome mutations in pre-meiotic germ cells(spermatogonia) in a random pattern.
UV-treatment can break down sperm-DNA; giving rise to haploid progenies(with only mothers DNA copy)(can live up to three days).
Heat shock-treatment of eggs during the first cell-cycle inhibits the first mitotic division -> diploid progeny
Transient transgene expression
mRNA transcript injection, stabilized with poly-A tails.
A) Inject into yolk of one cell -> whole body expression
B) Inject into one cell of the 8-cell stage embryo -> mosaicism
Transgenic fish generation
inject transposase gene mRNA + plasmid with EGFP. Flanked by Tol2 elements positioned at the 5’ and 3’ end of a minimal promoter + EGFP. Fish are raised to screen for founders -> have germline that can be transmitted to the next generation.
Gene knockdown by morpholino
oligomer molecule used to modify gene expression by binding to complementary sequence of RNA or SSDNA, they act as steric blockling. Injected in the 1-4 cell stage.
Gene knockout by CRISPR/Cas9
gRNA(tracrRNA+crRNA) is used together with Cas9 protein, they form a Cas9:gRNA complex -> binds to PAM sequence at target gene -> Cas9 makes ds-cut in DNA to disrupt gene. Can be used for insertion or deletion of bp:s or frameshifts.
In zebrafish the complex can be injected into the yolk of the embryo, 1 cell stage.
Xenograft
Graft of tissue taken from a donor of one species and grafted into a recipient of another species. Takes long time, high costs. Ex: Human tumour cells -> inject in mouse -> human tumor xenograft.
Cancer research
In zebrafish there are several pros:
no immunorejection in early embryo
easy to visualize the cancer cells due to its transparent body
quick analys
lower costs.
Studies demonstrate that human cancer cells actively invade within a zebrafish microenvironment, and form metastatic tumors at secondary tissue sites, suggesting that the mechanisms involved during the different stages of metastasis are conserved between humans and zebrafish, supporting the use of zebrafish embryos as a suitable/viable model of human cancer metastasis.
Tumor growth and spread requires angiogenesis(formation of new blood vessels). Hypoxia(low-oxygen) induces expression of VEGF(vascular endothelial growth factor), which stimulates angiogenesis.
