functional genomic studies (knock-down of gene function)

This study is based on the analysis of the loss of function of specific genes by the construction of a zebrafish with a temporary block of those specific genes (knock-down). With this purpose, block of mRNA translation can be performed as well as nuclear processing, more specifically pre-mRNA processing. To carry out both types of blocking, systems antisense oligonucleotides known as morpholinos (GeneTools LLC.), will be used. These morpholinos are specifically designed by ZF BioLabs, based on the sequence of the gene to be knocked down. These oligos will be microinjected in 2-8 cell zebrafish embryos, after selecting the best spawns. Microinjection can be also carry out in other stages depending on the stage of development of zebrafish embryo in which the gene of interest is expressed.

Once the gene expression is blocked, microinjected embryos will be analyzed. A phenotypical study will be performed in order to check the genes loss of function. This study involves an individual visualization of the zebrafish microinjected embryos previously placed in 96 well plates and the analysis of the different end-points including morphological, functional, lethal and embryo growth features.

To confirm the efficient block of the specific gene, Western Blot analysis will be carried out for mRNA translation block and RT-PCR for alternative splicing processing block.