9. VL

Question 1

which cascade is active during optic cup morphogenesis?

Answer 1

The Hippo kinase signaling

Optic cup morphogenesis defects in an Hippo signaling mutant

Question 2

Methods for transgenesis in zebrafish (3)

Answer 2

1. injection of capped mRNA
   • (A) EGFP mRNA is injected at the one-cell stage.
   • (B) EGFP mRNA is injected at the 8-cell stage.
   • (C) mRNA encoding for EGFP followed by the 3’ nanos UTR is injected at the one cell stage.
2. in section of an expression plasmid
3. Tol2 Transposon-mediated transgenesis
   (The DNA construct with two minimal Tol2 elements at the 3’ and 5’ flanking sites and the mRNA encoding for Tol2 transposase are injected into the first cell. Founder fish have germ cells with a construct integrated into the genome. The progeny derived from these transgenic germ cells in the F1 will have the tissue specific expression.)
   (A) Cre-induced recombination induces the loss of mCherry.
   (B) MAZe transgenic system. The construct with Gal4 and the EF1 promoter is ubiquitously expressed. Cre recombinase is under the control of heat shock promoter (Hsp70). Cre expression in a mosaic fashion drives red fluorescent protein (RFP). A construct expressed in the specific tissue allows the tissue- specific expression of additional proteins.

Question 3

Gene silencing by short hairpin RNAs

Answer 3

The miR30 gene transcribed region can be used to produce RNA hairpins, where the normal miR30 hairpin is replaced with a hairpin targeting a gene of interest.
shRNA is most efficiently to expressed from a promoter. Where the guide strand and target are exact matches, the target RNA is cleaved and subsequently degraded by endogenous nucleases.

Question 4

Knockout technologies in zebrafish

Answer 4

Zinc finger nucleases
The scheme shows the principle of the zinc finger nuclease mechanism of action. Each monomer of a ZFP recognizes a specific DNA sequence (labeled in blue and red), while the nuclease domains (FokI, labeled in orange) cleave the DNA. ZFN against the no tail gene were used.

Question 5

You can use CRISPR Cas9 for….

Answer 5

to knock-out genes, Spatiotemporal control of gene disruption with CRISPR-Cas9, Genome editing in zebrafish using the CRISPR/Cas9 system (homology-dependent repair), CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair, Intron targeting-mediated and endogenous gene integrity- maintaining knock-in using the CRISPR/Cas9 system
Perspectives: Edited cDNA insertions via homology-independent repair mechanisms or RNA targeting by CRISPR Cas13, Programmable RNA Tracking in Live Cells with CRISPR/Cas9

Question 6

Genom Engeneering by Crispr Cas

Answer 6

(A) Breaking Single-strand DNA
(B) Improving on-target Double-strand break specificity
(C) Generation of cellular models
(D) Generation of transgenic animal models
(E) Somatic genetic modification, obviating the need for embryonic manipulation
(F) Genome-scale functional screening
(G) Transcriptional regulation: direct transcriptional modulation or epigenetic control
(H) Live imaging of DNA, to understand how the structural organization of genome modulates functional output
(I) Temporal control of dynamic cellular processes