BACKGROUND
in vivo gene delivery into skeletal or cardiac muscle by direct injection of naked dna is a convenient method to express proteins  <cit> .
the efficiency of this procedure was improved substantially by applying electrical pulses at the site of injection  <cit> .
electroporation enhances transgene expression in tissues by causing electrical breakdown of membranes combined with electrophoresis of dna into cells  <cit> .
since its discovery, electrotransfer of dna into muscle has become a very popular method of gene delivery due to easy access of the muscle tissue, long life span of the muscle cell, abundant blood supply and its suitability for the production of proteins as systemic therapeutic agents  <cit> .
electroporation is routinely applied to a portion of the muscle, leading to transfection of cell layers around the site of injection.
recently, in ovo or in utero electroporation into the neural tubes and electroporation in tissue explants or solid tumor has provided promising results  <cit> .
zebrafish has proven to be a useful vertebrate complement to the mouse  <cit> .
easy husbandry, large supply of eggs and transparent early stages of development make zebrafish a convenient model organism to study vertebrate development  <cit> .
transgenic zebrafish have been routinely produced to understand the gene function by injecting the transgene into the eggs and following its expression in cell lineages  <cit> .
methods for large-scale generation of transgenic zebrafish were explored  <cit> .
electroporation was successfully demonstrated on dechorinated eggs of three fish species  <cit> .
recently, electroporation of the neural tube of the embryo was demonstrated to investigate gene regulation in later stages of zebrafish development  <cit> .
methods to express transgenes in adult fish are recently being investigated.
muscular injection of naked dna, particle bombardment using gene gun and electroporation of the fish fin  <cit>  after dna injection into the muscle.
electroporation using forceps electrodes placed on either side of the body of a fish resulted in substantial enhancement of reporter gene expression.
RESULTS
microinjection of naked dna into the fish muscle was sufficient for expression of transgene in muscle cells  <cit> .
to improve the efficiency of transgene expression further, we have attempted electroporation subsequent to muscle injection.
tweezer type electrodes were used to apply pulses by holding the sides of the fish between the electrode faces.
electroporation parameters, such as number of pulses, pulse duration, voltage strength and shape of the pulse have critical bearing on the transfection efficiency  <cit> .
each of these parameters is required to be optimized for each tissue since the biochemical and physical disposition of tissues to electroporation is known to be different  <cit> .
in all our experiments, square pulses of 60-ms duration were used.
initially we monitored the dependence of number of pulses on reporter gene expression at a fixed voltage of  <dig> v·cm- <dig>  luciferase activity was maximum at  <dig> pulses and decreased at lesser or higher number of pulses.
the maximal expression of reporter gene was nearly ten fold higher than that obtained without electroporation .
next, we investigated the effect of voltage on transgene expression by keeping the number of pulses constant.
voltage applied at  <dig> v·cm- <dig> was found to maximize the expression of reporter gene with  <dig> pulses .
the bell shaped dependency of reporter gene expression on number of pulses and voltage applied suggests that there is a significant influence of these parameters on cell entry of dna.
in addition to the electroporation parameters, the amount of plasmid dna used for electroporation is known to have strong bearing on the reporter gene expression  <cit> .
at  <dig> μg of plasmid dna per fish, we see significant increase in reporter gene expression on electroporation and above  <dig> μg of plasmid dna there is no further change in the gene expression .
below  <dig> μg of plasmid dna, we could not detect luciferase activity.
in an earlier report muscle injection of plasmid dna without electroporation showed maximum expression at  <dig> μg of dna into adult zebrafish  <cit> .
we have also tested these electroporation conditions on another fish species, indian carp .
six pulses at  <dig> v·cm- <dig> increased the reporter gene expression in the indian carp by more than eight fold  indicating that electroporation enhances reporter gene activities in the indian carp also .
transient gene expression from different promoters in zebrafish  and in a <dig> cell lines
to compare the strength of promoters on reporter gene expression in adult zebrafish and cell lines, we have constructed three plasmids each containing cmv  or human ef-1α  or xenopus ef-1α  promoter with luciferase as the reporter gene.
strength of promoters was tested in a <dig> cell lines after transfecting the cells with each of the plasmids using commercial lipofecting agent, lipofectamine .
pcmv-luc and pesg-luc showed equal reporter gene activity in a <dig> cells and it was 10-fold higher than pbos-luc.
we have estimated the reporter gene activity under the control of these three promoters in adult zebrafish using electroporation conditions described earlier i.e.,  <dig> pulses of  <dig> ms duration at  <dig> v·cm- <dig> with  <dig> μg of plasmid .
we plotted reporter gene activities obtained with the three plasmids in adult zebrafish along with the activities obtained at 1: <dig> charge ratio in a <dig> cells lines.
the relative strengths of the promoter were similar in zebrafish and in a <dig> cell lines.
in zebrafish cmv and xenopus ef-1α promoters showed 2– <dig> fold higher reporter gene activity compared to human ef-1α promoter .
ef-1α promoter derived from an amphibian demonstrates higher strengths for expression than the human homologue in zebrafish.
however, among the three promoters the folds increase in luciferase expression with human ef-1α promoter seems to be stronger in zebrafish than a <dig> cell lines.
in an earlier study on transient expression by naked dna injection into the muscle of adult zebrafish, cmv promoter was shown to be more efficient than sv  <dig> promoter.
in the same study the expression observed in muscle tissue of adult zebrafish was similar to the expression in rth- <dig> and rtg- <dig> cell lines  <cit> .
these experiments suggest that in vivo transient gene expression using electroporation in adult zebrafish would be a convenient way to study the strengths of promoters.
expression of gfp in zebrafish on electroporation
we investigated the histological expression of gfp upon electroporation of pcmvgfp in zebrafish.
the fish were killed two days after injection and the frozen section of the mid-, head- and tail region were taken .
in the absence of electroporation, green fluorescence was faintly visible in the tissue at the site of injection .
fluorescence was undetectable in control fish, which did not receive any injection.
significant gfp fluorescence was observed at the site of injection  upon electroporation .
in addition to the mid-region we could also observe gfp fluorescence, however less extensive, in tail and head region of the fish.
we have estimated the number of gfp positive cells from the phase contrast and nuclear stained  images of the same sections.
based on 50– <dig> cells per section and sections from three different fish, we observed 8%, 10% and 6% gfp positive cells in head-, mid- and tail-region, respectively, in non-electroporated fish.
the gfp positive cells increased to 15%, 70% and 25% in head, mid- and tail-region, respectively, upon electroporation.
higher expression of gfp in electroporated samples confirms the enhanced luciferase activities observed in fig.
<dig>  in a related study, particle bombardment in adult zebrafish resulted in gfp expression was observed in skin epithelial cells, muscle cells, neuron-like cells etc., whereas muscle injection resulted in transfection of several muscle bundles  <cit> .
appearance of fluorescence far from the site of injection is intriguing.
it may be possible that immediately after the injection the dna accesses other tissues before the application of pulses.
we observed that average time between muscle injection and electroporation was  <dig> s. electroporation affects the permeability of cells away from the site of injection and thereby enhances transgene expression in those tissues.
in addition the diameter of the disc on the tweezer type electrode is  <dig> mm may electroporate a larger area of the fish.
sudha et.al reported expression of gfp in non-muscle cells when the injections were made in the muscle  <cit> .
electroporation in zebrafish was used extensively on eggs and was also used for the functional analysis of the role of developmental genes in the neural tube of zebrafish embryo and larvae  <cit> .
electroporation was employed on the dechorinated zebrafish eggs and nearly 25% of fish showed transgene expression  <cit> .
muscle injection of dna and particle bombardment using gene gun were attempted in adult zebrafish and persistent gfp expression for  <dig> days was demonstrated  <cit> .
electroporation of fins is the only one reported case of electroporation in adult zebrafish.
fins were electroporated to deliver plasmid carrying gene for gfp and also used as a method to disrupt fgf signaling pathway during the process of regeneration  <cit> .
enhanced transgene expression upon electroporation in adult zebrafish would be useful in expression of proteins in the muscle and also in investigating tissue-specific expression of promoters and dna vaccination.
CONCLUSIONS
simplicity of muscular injection and application of electroporation on the zebrafish offers a protocol to achieve high expression of transgenes.
low voltage conditions, availability of commercial tweezer electrodes and safety of the procedure on fish allows to test the in vivo properties of regulatory gene sequences.
in addition to muscular injection of dna, electroporation would require an additional minute to administer.
observed correlation between in vivo  and in vitro  transfection efficiencies with three plasmid constructs suggests that in vivo transfections could be routinely performed in zebrafish.
high level of gene expression in adult zebrafish may offer a simple experimental system for functional analysis of gene sequences in zebrafish.

 in vivo gene delivery into skeletal or cardiac muscle by direct injection of naked dna is a convenient method to express proteins. the efficiency of this procedure was improved substantially by applying electrical pulses at the site of injection. electroporation enhances transgene expression in tissues by causing electrical breakdown of membranes combined with electrophoresis of dna into cells. since its discovery, electrotransfer of dna into muscle has become a very popular method of gene delivery due to easy access of the muscle tissue, long life span of the muscle cell, abundant blood supply and its suitability for the production of proteins as systemic therapeutic agents. electroporation is routinely applied to a portion of the muscle, leading to transfection of cell layers around the site of injection. recently, in ovo or in utero electroporation into the neural tubes and electroporation in tissue explants or solid tumor has provided promising results. zebrafish has proven to be a useful vertebrate complement to the mouse. easy husbandry, large supply of eggs and transparent early stages of development make zebrafish a convenient model organism to study vertebrate development. transgenic zebrafish have been routinely produced to understand the gene function by injecting the transgene into the eggs and following its expression in cell lineages. methods for large-scale generation of transgenic zebrafish were explored. electroporation was successfully demonstrated on dechorinated eggs of three fish species. recently, electroporation of the neural tube of the embryo was demonstrated to investigate gene regulation in later stages of zebrafish development. methods to express transgenes in adult fish are recently being investigated. muscular injection of naked dna, particle bombardment using gene gun and electroporation of the fish fin after dna injection into the muscle. electroporation using forceps electrodes placed on either side of the body of a fish resulted in substantial enhancement of reporter gene expression.  microinjection of naked dna into the fish muscle was sufficient for expression of transgene in muscle cells. to improve the efficiency of transgene expression further, we have attempted electroporation subsequent to muscle injection. tweezer type electrodes were used to apply pulses by holding the sides of the fish between the electrode faces. electroporation parameters, such as number of pulses, pulse duration, voltage strength and shape of the pulse have critical bearing on the transfection efficiency. each of these parameters is required to be optimized for each tissue since the biochemical and physical disposition of tissues to electroporation is known to be different. in all our experiments, square pulses of 60-ms duration were used. initially we monitored the dependence of number of pulses on reporter gene expression at a fixed voltage of v·cm- luciferase activity was maximum at pulses and decreased at lesser or higher number of pulses. the maximal expression of reporter gene was nearly ten fold higher than that obtained without electroporation. next, we investigated the effect of voltage on transgene expression by keeping the number of pulses constant. voltage applied at v·cm- was found to maximize the expression of reporter gene with pulses. the bell shaped dependency of reporter gene expression on number of pulses and voltage applied suggests that there is a significant influence of these parameters on cell entry of dna. in addition to the electroporation parameters, the amount of plasmid dna used for electroporation is known to have strong bearing on the reporter gene expression. at μg of plasmid dna per fish, we see significant increase in reporter gene expression on electroporation and above μg of plasmid dna there is no further change in the gene expression. below μg of plasmid dna, we could not detect luciferase activity. in an earlier report muscle injection of plasmid dna without electroporation showed maximum expression at μg of dna into adult zebrafish. we have also tested these electroporation conditions on another fish species, indian carp. six pulses at v·cm- increased the reporter gene expression in the indian carp by more than eight fold indicating that electroporation enhances reporter gene activities in the indian carp also. transient gene expression from different promoters in zebrafish and in a cell lines to compare the strength of promoters on reporter gene expression in adult zebrafish and cell lines, we have constructed three plasmids each containing cmv or human ef-1α or xenopus ef-1α promoter with luciferase as the reporter gene. strength of promoters was tested in a cell lines after transfecting the cells with each of the plasmids using commercial lipofecting agent, lipofectamine. pcmv-luc and pesg-luc showed equal reporter gene activity in a cells and it was 10-fold higher than pbos-luc. we have estimated the reporter gene activity under the control of these three promoters in adult zebrafish using electroporation conditions described earlier i.e., pulses of ms duration at v·cm- with μg of plasmid. we plotted reporter gene activities obtained with the three plasmids in adult zebrafish along with the activities obtained at 1: charge ratio in a cells lines. the relative strengths of the promoter were similar in zebrafish and in a cell lines. in zebrafish cmv and xenopus ef-1α promoters showed 2– fold higher reporter gene activity compared to human ef-1α promoter. ef-1α promoter derived from an amphibian demonstrates higher strengths for expression than the human homologue in zebrafish. however, among the three promoters the folds increase in luciferase expression with human ef-1α promoter seems to be stronger in zebrafish than a cell lines. in an earlier study on transient expression by naked dna injection into the muscle of adult zebrafish, cmv promoter was shown to be more efficient than sv promoter. in the same study the expression observed in muscle tissue of adult zebrafish was similar to the expression in rth- and rtg- cell lines. these experiments suggest that in vivo transient gene expression using electroporation in adult zebrafish would be a convenient way to study the strengths of promoters. expression of gfp in zebrafish on electroporation we investigated the histological expression of gfp upon electroporation of pcmvgfp in zebrafish. the fish were killed two days after injection and the frozen section of the mid-, head- and tail region were taken. in the absence of electroporation, green fluorescence was faintly visible in the tissue at the site of injection. fluorescence was undetectable in control fish, which did not receive any injection. significant gfp fluorescence was observed at the site of injection upon electroporation. in addition to the mid-region we could also observe gfp fluorescence, however less extensive, in tail and head region of the fish. we have estimated the number of gfp positive cells from the phase contrast and nuclear stained images of the same sections. based on 50– cells per section and sections from three different fish, we observed 8%, 10% and 6% gfp positive cells in head-, mid- and tail-region, respectively, in non-electroporated fish. the gfp positive cells increased to 15%, 70% and 25% in head, mid- and tail-region, respectively, upon electroporation. higher expression of gfp in electroporated samples confirms the enhanced luciferase activities observed in fig. in a related study, particle bombardment in adult zebrafish resulted in gfp expression was observed in skin epithelial cells, muscle cells, neuron-like cells etc., whereas muscle injection resulted in transfection of several muscle bundles. appearance of fluorescence far from the site of injection is intriguing. it may be possible that immediately after the injection the dna accesses other tissues before the application of pulses. we observed that average time between muscle injection and electroporation was s. electroporation affects the permeability of cells away from the site of injection and thereby enhances transgene expression in those tissues. in addition the diameter of the disc on the tweezer type electrode is mm may electroporate a larger area of the fish. sudha et.al reported expression of gfp in non-muscle cells when the injections were made in the muscle. electroporation in zebrafish was used extensively on eggs and was also used for the functional analysis of the role of developmental genes in the neural tube of zebrafish embryo and larvae. electroporation was employed on the dechorinated zebrafish eggs and nearly 25% of fish showed transgene expression. muscle injection of dna and particle bombardment using gene gun were attempted in adult zebrafish and persistent gfp expression for days was demonstrated. electroporation of fins is the only one reported case of electroporation in adult zebrafish. fins were electroporated to deliver plasmid carrying gene for gfp and also used as a method to disrupt fgf signaling pathway during the process of regeneration. enhanced transgene expression upon electroporation in adult zebrafish would be useful in expression of proteins in the muscle and also in investigating tissue-specific expression of promoters and dna vaccination.  simplicity of muscular injection and application of electroporation on the zebrafish offers a protocol to achieve high expression of transgenes. low voltage conditions, availability of commercial tweezer electrodes and safety of the procedure on fish allows to test the in vivo properties of regulatory gene sequences. in addition to muscular injection of dna, electroporation would require an additional minute to administer. observed correlation between in vivo and in vitro transfection efficiencies with three plasmid constructs suggests that in vivo transfections could be routinely performed in zebrafish. high level of gene expression in adult zebrafish may offer a simple experimental system for functional analysis of gene sequences in zebrafish. 
electroporation parameters such as number of pulses, voltage and amount of plasmid dna were optimized and it was found that pulses of v·cm- at μg of plasmid dna per fish increased the luciferase expression 10-fold compared to controls. electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. in this study we investigated the effect of electroporation on expression of transgenes in 3– month old adult zebrafish. to establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in a cells and adult zebrafish after electroporation. the relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. electroporation after dna injection enhances gene expression 10-fold in adult zebrafish. application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish. similar enhancement in transgene expression was also observed in indian carp.
 expression of transgenes in muscle by injection of naked dna is widely practiced. application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. zebrafish is a precious model to investigate developmental biology in vertebrates. in this study we investigated the effect of electroporation on expression of transgenes in 3– month old adult zebrafish.  electroporation parameters such as number of pulses, voltage and amount of plasmid dna were optimized and it was found that pulses of v·cm- at μg of plasmid dna per fish increased the luciferase expression 10-fold compared to controls. similar enhancement in transgene expression was also observed in indian carp. to establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in a cells and adult zebrafish after electroporation. the relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. gfp fluorescence in tissues after electroporation was also studied by fluorescence microscopy.  electroporation after dna injection enhances gene expression 10-fold in adult zebrafish. electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish. 