Haiyan Wan

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.

Expression analyses of zebrafish transferrin, ifabp, and elastaseB mRNAs as differentiation markers for the three major endodermal organs: liver, intestine, and exocrine pancreas.

In the present work, three zebrafish cDNA clones encoding transferrin, intestinal fatty acid binding protein (IFABP), and elastaseB were cloned and their expression patterns in early zebrafish development were characterized as differentiation markers for the three major endoderm organs: liver, intestine, and exocrine pancreas. transferrin and ifabp mRNAs exhibit a biphasic expression pattern during early development. transferrin mRNAs were first expressed at ∼7 hours postfertilization (hpf) in the yolk syncytial layer (YSL) and later in the liver rudiment (from ∼48 hpf) and in the esophagus transiently (72–96 hpf). Ifabp mRNAs were initially expressed in the YSL at the ventral side during late epiboly (8–9 hpf), spread throughout the YSL of later stage embryos, and appeared in the intestine rudiment at ∼36 hpf. In contrast to the transferrin and ifabp mRNAs, elastaseB mRNAs were not expressed in the yolk sac or YSL, and these transcripts were detected exclusively in the exocrine pancreas after ∼56 hpf. Developmental Dynamics 230:165–173, 2004.

Green fluorescent protein (GFP) transgenic fish and their applications

The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.

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.

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.

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.