Editing Zebrafish (section)

====Gene expression====
Due to their fast and short life cycles and relatively large clutch sizes, ''D. rerio'' or zebrafish are a useful model for genetic studies. A common [[reverse genetics]] technique is to [[gene knockdown|reduce gene expression]] or modify [[Splicing (genetics)|splicing]] using [[Morpholino]] [[antisense]] technology. Morpholino [[oligonucleotide]]s (MO) are stable, synthetic [[macromolecule]]s that contain the same [[nucleoside|bases]] as DNA or RNA; by binding to complementary RNA sequences, they can reduce the [[gene expression|expression]] of specific genes or block other processes from occurring on RNA. MO can be injected into one cell of an embryo after the 32-cell stage, reducing gene expression in only cells descended from that cell. However, cells in the early embryo (less than 32 cells) are permeable to large molecules,<ref name=blast2>{{cite journal |vauthors=Kimmel CB, Law RD |title=Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells |journal=Developmental Biology |volume=108 |issue=1 |pages=78–85 |date=March 1985 |pmid=3972182 |doi=10.1016/0012-1606(85)90010-7}}</ref><ref name=blast4>{{cite journal |vauthors=Kimmel CB, Law RD |title=Cell lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and gastrula stages |journal=Developmental Biology |volume=108 |issue=1 |pages=94–101 |date=March 1985 |pmid=3972184 |doi=10.1016/0012-1606(85)90012-0}}</ref> allowing diffusion between cells. Guidelines for using Morpholinos in zebrafish describe appropriate control strategies.<ref>{{cite journal |vauthors=Stainier DY, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SC, Meng A, Mochizuki N, Panula P, Moens CB |display-authors=6 |title=Guidelines for morpholino use in zebrafish |journal=PLOS Genetics |volume=13 |issue=10 |pages=e1007000 |date=October 2017 |pmid=29049395 |pmc=5648102 |doi=10.1371/journal.pgen.1007000 |doi-access=free}}</ref> Morpholinos are commonly [[microinjection|microinjected]] in 500pL directly into 1–2 cell stage zebrafish embryos. The morpholino is able to integrate into most cells of the embryo.<ref>{{cite journal |vauthors=Rosen JN, Sweeney MF, Mably JD |title=Microinjection of zebrafish embryos to analyze gene function |journal=Journal of Visualized Experiments |issue=25 |date=March 2009 |pmid=19274045 |pmc=2762901 |doi=10.3791/1115}}</ref>

A known problem with gene knockdowns is that, because the genome underwent a [[genome#Genome evolution|duplication]] after the divergence of [[ray-finned fish]]es and [[lobe-finned fish]]es, it is not always easy to silence the activity of one of the two gene [[paralog]]s reliably due to [[Complementation (genetics)|complementation]] by the other paralog.<ref>{{cite journal |vauthors=Leong IU, Lan CC, Skinner JR, Shelling AN, Love DR |title=In vivo testing of microRNA-mediated gene knockdown in zebrafish |journal=Journal of Biomedicine & Biotechnology |volume=2012 |page=350352 |year=2012 |pmid=22500088 |pmc=3303736 |doi=10.1155/2012/350352 |publisher=Hindawi |doi-access=free}}</ref> Despite the complications of the zebrafish [[genome]], a number of commercially available global platforms exist for analysis of both gene expression by [[expression profiling|microarrays]] and promoter regulation using [[ChIP-on-chip]].<ref>{{cite journal |vauthors=Tan PK, Downey TJ, Spitznagel EL, Xu P, Fu D, Dimitrov DS, Lempicki RA, Raaka BM, Cam MC |display-authors=6 |title=Evaluation of gene expression measurements from commercial microarray platforms |journal=Nucleic Acids Research |volume=31 |issue=19 |pages=5676–5684 |date=October 2003 |pmid=14500831 |pmc=206463 |doi=10.1093/nar/gkg763}}</ref>