Tie Yan

Rapid identification and isolation of zebrafish cDNA clones

A fast and economical approach, referred to as cDNA clone tagging, was adapted to identify and isolate zebrafish cDNA clones. The basic approach was to partially sequence the coding region of size selected cDNA clones and the partial sequences were then used as tags for identifying the clones through homology search. To benefit maximally from the tagging approach, two cDNA libraries, derived from embryonic and adult fish poly(A)+ RNAs, respectively, were constructed by unidirectional cloning; conceptually, they have the potential to represent all expressed zebrafish genes. A total of 1084 clones were sequenced from the two libraries, and 511 clones were identified, based on sequence homology. These identified clones were derived from at least 261 genes, encoding 48 translational machinery proteins, 47 cytosolic proteins, 43 cytoskeletal proteins, 41 nuclear proteins, 32 membrane proteins, 22 secreted proteins, 20 mitochondrial proteins and 8 proteins with an unknown location. Of the 261 distinct cDNA clones identified, 254 were isolated for the first time in the zebrafish. These tagged cDNA clones, identified and unidentified, provide rich resources for developmental analysis as well as mapping of zebrafish genome. The long-term objective of this study is to establish a tagged zebrafish gene library that can be accessed both by hybridization screening against the plasmid DNAs and by electronic screening using the sequence information.

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior‐ and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2‐kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8‐GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter

A 1,934‐bp muscle‐specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5′ promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis‐elements in the promoter and that a minimal −77‐bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2‐gfp constructs. GFP expression in the 1,934‐bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871‐bp promoter. Our observation indicates that there is no distinct cis‐element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP‐dependent protein kinase A was injected into mylz2‐gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP‐expressing fast muscle. The mylz2‐gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered. Developmental Dynamics 227:14–26, 2003.

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Abstract: Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F1 transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.

Generation of living color transgenic zebrafish

Use of green fluorescent protein (gfp) as a reporter gene is a powerful approach for the investigation of tissue-specific gene expression and cellular localization of proteins because the fluorescence of its protein product can be conveniently detected in living cells without supplementing a substrate. The approach is particularly useful in zebrafish because of the transparency and external development of their embryos. In the past few years, using several zebrafish tissue-specific promoters, we have developed several stable lines of gfp transgenic zebrafish that display green fluorescence in different tissues; these include five transgenic lines under an epidermis-specific keratin8 (krt8) promoter, two transgenic lines under a fast skeletal muscle-specific promoter from the myosin light polypeptide 2 (mylz2) gene, and five transgenic lines under an elastaseA (elaA) promoter that is specifically expressed in pancreatic exocrine cells. In all cases, transgenic GFP is faithfully expressed according to the specificity of the promoters used. These gfp transgenic lines are useful for recapitulation of a gene expression program, investigation of tissue and organ development, cell sorting for in vitro cell culture, and construction of cell type-specific cDNA library. Recently, by using two tissue-specific promoters linked to two different reporter genes, gfp and rfp (red fluorescent protein), we have generated two-color transgenic zebrafish that express GFP in skin epidermis and RFP in fast skeletal muscle. Currently, we are also developing gfp transgenic fish for biomonitoring using selected inducible gene promoters that can respond to heavy metals and estrogenic compounds. Thus, generation of living color transgenic zebrafish will have important applications in biotechnology as well as in developmental biology.