Molecular Biotechnology and Genetics
Marine Biotechnology
EJB Electronic Journal of Biotechnology ISSN: 0717-3458
© 2001 by Universidad Católica de Valparaíso -- Chile
BIP RESEARCH ARTICLE

Tilapia chromosomal growth hormone gene expression accelerates growth in transgenic zebrafish (Danio rerio)

Reynold Morales
Mammalian Cell Genetics Division
Center for Genetic Engineering and Biotechnology
PO Box 6162, Havana 10600, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: reynold.morales@cigb.edu.cu

María Teresa Herrera
Department of Animal and Human Biology
Faculty of Biology, University of Havana
25th street No. 455, Havana 10400, Cuba

Amílcar Arenal
Center for Genetic Engineering and Biotechnology
PO Box 387, Camagüey 1, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: amilcar.arenal@cigbcam.cigb.edu.cu

Asterio Cruz
Division of Quality Control and Assurance
Center for Genetic Engineering and Biotechnology
PO Box 6162, Havana, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: asterio.cruz@cigb.edu.cu

Oscar Hernández
Center for Genetic Engineering and Biotechnology
PO Box 387, Camagüey 1, Cuba

Rafael Pimentel
Center for Genetic Engineering and Biotechnology
PO Box 387, Camagüey 1, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: rafael.pimentel@cigbcam.cigb.edu.cu

Isabel Guillén
Mammalian Cell Genetics Division
Center for Genetic Engineering and Biotechnology
PO Box 6162, Havana, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: isabel.guillen@cigb.edu.cu

Rebeca Martínez
Mammalian Cell Genetics Division
Center for Genetic Engineering and Biotechnology
PO Box 6162, Havana, Cuba
Tel: 537-216022
Fax: 537-331779
E-mail: rebeca.martinez@cigb.edu.cu

Mario P Estrada*
Mammalian Cell Genetics Division
Center for Genetic Engineering and Biotechnology
PO Box 6162, Havana, Cuba
Tel: 537-218008 / 537-218466
Fax: 537-36008 / 537-218070
E-mail: mpablo@cigb.edu.cu

* Corresponding author

Keywords: carp, GFP, growth hormone, tilapia, transgenic, zebrafish.

BIP Article

Gene transfer technology has produced a great impact in modern biology and biotechnology (Powers et al. 1998). A number of fish species are in focus for gene transfer experiments and can be divided into two main groups: animals used in aquaculture (Fletcher and Davies, 1991; Hew et al. 1995; Chen and Lu, 1998) and model fish used in basic research (Chen and Lu, 1998). Among the major food fish species are carp (Cyprinus sp.), tilapia (Oreochromis sp.), salmon (Salmo sp., Oncorhynchus sp.) and channel catfish (Ictalurus punctatus) while zebrafish (Danio rerio), medaka (Oryzias latipes) and goldfish (Carassius auratus) are used in basic research.

Zebrafish is an already well-established model organism (Kimmel, 1989; Westerfield, 1995). This fish offers the possibility of combining rapid early development, which is amenable to direct observation and manipulation, large numbers of progeny from a single mating, and a relatively short generation time (2 to 3 months).

Transgenic technology through DNA microinjection into zebrafish embryos has made great gain in the last decade. Stuart et al. (1990) first showed that the DNA injected into the cytoplasm of fertilized zebrafish eggs could integrate into the fish genome and be inherited in the germ line. Culp et al. (1991) demonstrated that the frequency of germline transmission of a microinjected DNA could be as a high as 20% in zebrafish. To improve the efficiency of selection of transgenics, genetic markers are co-injected with the transgene to monitor for transformed zygotes. The green fluorescent protein (GFP) from Jellyfish (Aequorea victoria) has been used for this purpose in zebrafish (Amsterdam et al. 1995; Peters et al. 1995).

The results showed that GFP expression is a good indicator of stable transformation. Transgenic F1 zebrafish grew 20% faster than full sibling non-transgenic controls.

Materials and Methods

Detection of GFP gene expression

Following microinjection, GFP gene expression was followed in 24 h after microinjection embryos using a Zeiss (Germany) epifluorescence microscope (excitation 450-490 nm, barrier filter LP 520 nm). Photographs were taken using Kodak chrome 1200 asa films.

Analysis of growth performance

A random transgenic female zebrafish founder was crossed with a non-transgenic male to produce F1 progeny. Fifty F1 zebrafish of four week-old were randomly selected and grown individually under similar conditions of water temperature (28ºC) and photoperiod (10 h light: 14 h dark). Fish were fed 3 times daily with brine shrimp eggs and brine shrimp flake (Argent Chemical Laboratories, USA). Zebrafish were weighed weekly during 6 weeks to monitor growth performance. In the course of the experiment, fin DNA was extracted and assayed for transgene identification. Weight of transgenic and non-transgenic full siblings was compared employing a Student t-Test.

Results

Cloning of chrtiGH

The structure of the O. hornorum chrtiGH gene was similar to the structure reported for O. niloticus. At the nucleotide level, we found in the coding region a change of a guanine instead of an adenine in the position 594 of the O. niloticus tiGH cDNA (Ber and Daniel, 1992). However, the deduced aminoacid sequence of the O. hornorum tilapia GH was similar to the sequence reported for O. niloticus (Ber and Daniel, 1992).

Generation of transgenic zebrafish

Zebrafish embryos were collected and microinjected in several batches with an average of 250 (214-293) embryos per injection batch (Table 1). The survival rate 24 h post-injection averaged 62% (56-75%) (Table 1). Non-injected embryos showed a similar survival rate. GFP was monitored in 24 h post-injection embryos and fluorescence was detected in 1.2% (0.9-1.4%) of the injected embryos (Table 1). After PCR analysis of fin DNA, 0.8% (0.4-1.1%) of injected embryos resulted in transgenic fish (Table 1).

Zebrafish embryos were collected and microinjected with cbp-chrtiGH : pRSGFP (10:1 molar ratio). Survival and GFP fluorescence rates were assayed 24 h post-injection. Transgenic fish were screened for the presence of cbp-chrtiGH sequences by PCR analysis of fin DNA.

The GFP expression pattern in 24 h post-injection zebrafish embryos was patchy and in different regions of the embryo. Under our experimental conditions, GFP expression was a good indicator of embryo transformation as 67% (50-100%) of fluorescent embryos resulted in fish positive for transgene sequences after PCR analysis.

A transgenic female was selected for further characterization and studies. Southern blot analysis of fin DNA indicated that the transgene was present with a size corresponding to the injected fragment (Figure 1). This female was used as P1 founder to obtain F1 descendants after crossing to a non-transgenic male. The transgene was transmitted to 46% of F1 fish.

Characterization of the growth phenotype in transgenic zebrafish

For analysis of growth performance, F1 transgenic and full sibling non-transgenic control fish were grown under similar conditions and weighed weekly during 6 weeks. Transgenic zebrafish grew faster than controls (Figure 2). At the start of the experiment, the weight (mean ± SD) of transgenics (0.12 ± 0.04 g) and controls (0.11 ± 0.04 g) was similar (P = 0.2, Student t-Test). Six weeks later, transgenic fish were 20% heavier than controls (0.36 ± 0.10 g vs. 0.31± 0.08 g; P = 0.03, Student t-Test). The increment in weight for the period of study was also statistically significant (P = 0.04, Student t-Test) between transgenic (0.24 ± 0.09 g) and control (0.20 ± 0.07 g) fish.

References

Amsterdam A.; Lin S.; Moss L.G. and Hopkins N. (1995). Requirements for green fluorescent protein detection in transgenic Zebrafish embryos. Gene 173:99-103.

Ber R. and Daniel V. (1992). Structure and sequence of the growth hormone encoding gene from Tilapia nilotica. Gene 113:245-250.

Chen T.T. and Lu J-K. (1998). Transgenic fish technology: Basic principles and its application in basic and applied research. In: de la Fuente J. and Castro F.O. eds. Gene transfer in aquatic organisms. RG Landes Company and Germany: Springer-Verlag, Austin, Texas, USA. pp. 45-73

Culp P.; Nusslein-Volhard C. and Hopkins N. (1991). High frequency germ-line transmission of plasmid DNA sequences injected into fertilized Zebrafish eggs. Proceedings of the National Academy of Sciences 88:7953-7957.

Fletcher G.L. and Davies P.L. (1991). Transgenic fish for aquaculture. Genetic Engineering 13:331-371.

Hew C.L.; Fletcher G.L. and Davies P.L. (1995). Transgenic salmon: tailoring the genome for food production. Journal of Fish Biology 47:1-19.

Kimmel C. (1989). Genetics and early development of Zebrafish. Trends Genetic Science 5:283-288.

Peters K.G.; Rao P.S.; Bell B.S. and Kindman L.A. (1995). Green fluorescent fusion proteins: powerful tools for monitoring protein expression in live zebrafish embryos. Developmental Biology 171:252-257.

Powers D.A.; Gómez-Chiarri M.; Chen T.T. and Dunham R. (1998). Genetic Enginering of Finfish and shellfish. In: de la Fuente J. and Castro F.O. eds. Gene transfer in aquatic organisms. RG Landes Company and Germany, Springer-Verlag, Austin, Texas, USA. pp. 17-34.

Westerfield M. (1995). The Zebrafish Book. A guide for the laboratory use of zebrafish (Brachydanio rerio). University of Oregon Press. Eugene, Oregon.

Supported by UNESCO / MIRCEN network
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