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A transgene targeted to the zebrafish nkx2.4b locus drives specific green fluorescent protein expression and disrupts thyroid development
Author(s) -
Hutcheson David A.,
Xie Yuanyuan,
Figueroa Priscilla,
Dorsky Richard I.
Publication year - 2020
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.224
Subject(s) - transcription activator like effector nuclease , biology , transgene , zebrafish , green fluorescent protein , exon , genetics , microbiology and biotechnology , mutant , locus (genetics) , gene , genome editing , genome
Abstract Background With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b , a gene expressed during hypothalamic and thyroid development. We combined transcription activator‐like effector nucleases (TALENs)‐mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus. Results Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes. Conclusions By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss‐of‐function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development.