Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis

Abstract

The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4-5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.

The origins and evolution of sleep

“…While identifying sleep in jellyfish and molluscs opens the door for studies for sleep function, rhythmic circadian behavior or gene expression has been identified in many other systems that have not been analysed for sleep (Joiner, 2016). For example, the starlet sea anemone Nematostella vectensis has robust circadian behavioral and molecular rhythms, a simplified and transparent nervous system, and is amenable to transgenic manipulation (Hendricks et al, 2012;Peres et al, 2014;Oren et al, 2015;Renfer and Technau, 2017). Molecular circadian rhythms have also been identified in the tunicate sea squirt Ciona intestinalis that are accompanied by circadian-dependent regulation of oxygen consumption (Minamoto et al, 2010).…”

NvPOU4/Brain3functions as a terminal selector gene in the nervous system of the cnidarianNematostella vectensis

“…The NvPOU4::memGFP transgenic reporter line was generated by meganuclease-mediated transgenesis as described by (Renfer and Technau, 2017 (2)::mOrange line has been described in (Richards and Rentzsch, 2014), NvElav1::mOrange in (Nakanishi et al, 2012), NvNCol3::mOrange in (Sunagar et al, 2018) and NvFoxQ2d::mOrange in (Busengdal and Rentzsch, 2017). An overview of the crosses are provided in Table S4.…”

Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis

“…Since the early 2000s, Nematostella researchers have developed several protocols including maintenance and spawning procedures 14,15 , isolation of protein and nucleic acids 16 , in situ hybridization 17, ,, 18 , microinjection and morpholino-based gene knockdown 19 , meganuclease-mediated transgenesis 20 and CRISPR/Cas9-based gene editing 21 . Recently, we developed a gene knockdown method based on microinjection of gene-specific shRNA into Nematostella eggs (He et al, in press 2018).…”