Electroporation of salmon sperm for gene transfer: Efficiency, reliability, and fate of transgene

Sin, F. Y. T.; Walker, Seumas P.; Symonds, Jane E.; Mukherjee, Urmi; Khoo, Jenny; Sin, Iris L.

doi:10.1002/(sici)1098-2795(200006)56:2+<285::aid-mrd16>3.0.co;2-4

Cited by 43 publications

(14 citation statements)

References 11 publications

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“…PCR analysis of the presumptive P 1 transgenic fish showed that about 30 % of the presumptive P 1 transgenic animals carried the cecropin P1 transgene and 25 % carried the CF-17 transgene. These results are consistent with those reported by other investigators (Powers et al 1992; Symonds et al 1994; Sin et al 2000; Lu et al 2002). A total of eight F 1 founder heterozygous transgenic families carrying cecropin P1 transgene and nine F 1 heterozygous families carrying CF-17 transgene were established, respectively.…”

Section: Discussionsupporting

confidence: 93%

Production of Homozygous Transgenic Rainbow Trout with Enhanced Disease Resistance

et al. 2013

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Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F3 heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.

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Section: Discussionsupporting

confidence: 93%

Production of Homozygous Transgenic Rainbow Trout with Enhanced Disease Resistance

et al. 2013

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“…Sperm mediated gene transfer has been successfully applied in mammals [7][8][9], birds [10], amphibians [11] and some invertebrates [12,13]. Good SMGT results for fish were obtained only when semen was electroporated, as demonstrated for tilapia Oreochromis niloticus [14], zebrafish Danio rerio [15], salmon Oncorhynchus tshawytscha [16], grass carp Ctenopharyngodon idellus [17], and silver sea bream Sparus sarba [3]. However, if semen was incubated with exogenous DNA but not electroporated, the efficiency of SMGT for transgenic fish production was low or nonexistent [17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus

2009

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Sperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/10(6) cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa; controlling this activity improved the efficiency of SMGT in fish.

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“…The efficiency of SMGT depends on sperm viability and progressive motility (Lavitrano et al 2006). Foreign DNA uptake by spermatozoa has been studied in many species like ram (Castro et al 1991), bull (Atkinson et al 1991, cattle and chicken (Shemesh et al 2000), salmon (Sin et al 2000), shell fish (Tsai 2000), zebrafish (Khoo 2000), pigs (Lavitrano et al 2002) and rabbit .…”

Section: Introductionmentioning

confidence: 99%

Uptake of exogenous bovine GH–pmKate2–N expression vector by rams spermatozoa

et al. 2019

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Background: Sperm-mediated gene transfer (SMGT) is a technique that utilizes the ability of the spermatozoa to take up exogenous DNA. Growth hormone is anabolic hormone that plays an important role in muscle-building process and milk production in all animals. High blood concentration of growth hormones (GH) was observed for animals that were genetically selected for high milk production or for low carcass fatness levels. The present study aimed to investigate and enhance the capacity of ovine spermatozoa to uptake exogenous growth hormone cDNA and its impact on sperm motility. The current study is an introduction for further future studies to produce transgenic Egyptian sheep characterized with high productive performance. Methods: The growth hormone cDNA sequence was extracted from pituitary gland of Egyptian × Holstein (EH_ GH) cattle and subcloned into the pmKate2-N vector to construct the EH_GH-pmKate2-N expression vector. The complete sequence of EH_GH mRNA was registered in GenBank (AC: KP221576). A total of three groups were assessed for the sperm uptake experiment, namely, negative control, positive control, and dimethyl sulfoxide (DMSO) groups; all treated groups were incubated with the EH_GH-pmKate2-N vector. The expression of EH_GH protein was detected in DH10B cells using a fluorescence microscope and the SDS polyacrylamide gel electrophoresis. Results: The EH_GH-pmKate2-N vector was expressed in cultured Escherichia coli cells, and the molecular weight of EH_GH protein was 24,558 Da. The EH_GH-pmKate2-N vector was introduced efficiently into the heads of the spermatozoa in the DMSO and positive control groups. Incubation of the spermatozoa with the vector caused a significant reduction in progressive motility compared to the negative control. Conclusion: The present results demonstrated the ability of ovine spermatozoa to take up the exogenous vector without notable deleterious effects on sperm motility. In subsequent studies, the successful introduction of the exogenous GH expression vector into the sperm head allows for the production of GH-transgenic sheep characterized by a high growth rate in order to reduce the meat shortage in Egypt.

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Electroporation of salmon sperm for gene transfer: Efficiency, reliability, and fate of transgene

Cited by 43 publications

References 11 publications

Production of Homozygous Transgenic Rainbow Trout with Enhanced Disease Resistance

Production of Homozygous Transgenic Rainbow Trout with Enhanced Disease Resistance

Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus

Uptake of exogenous bovine GH–pmKate2–N expression vector by rams spermatozoa

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