Association of Foreign DNA with Sperm of Gilthead Seabream, Sparus aurata, After Sonication, Freezing, and Dimethyl Sulfoxide Treatments

Liu, X Y; Zohar, Yonathan; Knibb, Wayne

doi:10.1007/pl00011765

Cited by 5 publications

(5 citation statements)

References 22 publications

(22 reference statements)

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“…While the effect on initial cleavage was not consistent, bovine blastocyst development was significantly reduced after IVF with DMSO-treated sperm. This indicates some damaging effect of DMSO on sperm integrity and capacity to contribute towards normal development, similar to what has been described in fish (Liu et al 1999). This damage could be alleviated by the inclusion of lipofectamine (DM3/LP1 in Table 1).…”

Section: Discussionsupporting

confidence: 77%

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (nZ96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (nZ751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68Z56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (nZ48) or GFP fluorescence (nZ94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

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Section: Discussionsupporting

confidence: 77%

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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“…In the short±medium term, the prospects of developing commercially valuable ®sh lines with genetic engineering appear to be remote for various technical and social reasons, including the lack of ef®cient transformation vectors (Knibb 1997;Knibb, Moav & Elizur 1994, Knibb et al 1998aLiu, Zohar & Knibb 1999). In the short±medium term, the prospects of developing commercially valuable ®sh lines with genetic engineering appear to be remote for various technical and social reasons, including the lack of ef®cient transformation vectors (Knibb 1997;Knibb, Moav & Elizur 1994, Knibb et al 1998aLiu, Zohar & Knibb 1999).…”

Section: Genetic Engineeringmentioning

confidence: 99%

“…In the short–medium term, the prospects of developing commercially valuable fish lines with genetic engineering appear to be remote for various technical and social reasons, including the lack of efficient transformation vectors (Knibb 1997; Knibb, Moav & Elizur 1994, Knibb et al . 1998a; Liu, Zohar & Knibb 1999).…”

Section: Genetic Engineeringmentioning

confidence: 99%

Genetic improvement of marine fish - which method for industry?

Knibb

2000

Aquaculture Research

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Aquaculture industries and research organizations usually fail to exploit genetics as a tool for enhancing productivity and competitiveness. Yet, there is now some awareness among aquaculturists of the benefits from genetics. There remains little consensus about the types of genetic approaches that are most beneficial for the aquaculture industry. Discord arises because genetics comprises many subdisciplines, each with advocates promoting one field or another as the best method to develop commercially valuable strains. Here, I examine retrospectively the various genetic approaches in aquaculture to assess their commercial benefit.

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“…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]. The only exception was that Khoo et al [21] reported high rates of transgenic zebrafish production by incubating spermatozoa directly with exogenous DNA.…”

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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Association of Foreign DNA with Sperm of Gilthead Seabream, Sparus aurata, After Sonication, Freezing, and Dimethyl Sulfoxide Treatments

Cited by 5 publications

References 22 publications

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Genetic improvement of marine fish - which method for industry?

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

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