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Genetic tools for study of the hypothalamus and thyroid in Zebrafish
Author(s) -
Hutcheson David,
Xie Yuanyuan,
Duncan Robert,
Dorsky Richard
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.83.5
Subject(s) - transcription activator like effector nuclease , biology , genetics , homologous recombination , transgene , zebrafish , gene , genome , genome editing
The hypothalamus plays is a key regulatory role in a wide variety of animal behaviors including feeding, aggression, and mating. To further characterize the development and function of this CNS structure, we have begun to develop genetic tools with which to manipulate the hypothalamus in living zebrafish. Nkx2.4b is specifically expressed in the hypothalamus and thyroid during development, and is therefore an attractive target for genetic manipulation. First, we used TALENs to produce an Nkx2.4b −/− null. Damage caused by TALENs is typically repaired by non‐homologous end joining (NHEJ). We have obtained and bred several lines containing NHEJ in/dels that produce frameshifts in the 1 st exon of Nkx2.4b and are predicted to be null alleles. Second, we are testing the ability of TALENS to mediate a targeted homologous recombination (HR). We created a plasmid containing Nkx2.4b genomic sequence flanking a GFP transgene, such that homologous recombination would create a “knock‐in knock‐out”. This plasmid was co‐injected along with Nkx2.4b TALENS RNAs. Initial results suggest we have successfully inserted our construct, both as a random transgene insertion as well as in a targeted manner to the Nkx2.4b locus. Confirmation by genetic mapping is underway. In nulls, the hypothalamus appears normal while the thyroid shows complete loss of both developmental and functional markers. Additionally, we seek to develop a homologous recombination vector containing a fluorophore‐2A‐Cre ERT2 transgene flanked by Nkx2.4b genomic sequence. This will allow equimolar expression of both a fluorescent protein as well as the Cre ERT2 protein. Using the same strategy we used to insert the GFP transgene into the genome, we will create a Cre driver suitable for temporally controlled manipulation of hypothalamic cells. This tool will enable a wide range of studies with the potential to greatly enhance our knowledge of hypothalamic development and function. Support or Funding Information R01 NS082645 ‐ Regulation of hypothalamic radial glia by Wnt signaling