Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method
A high efficient and simple transgenic technology on mice and rabbits to transfect spermatozoa with exogenous DNA/DMSO complex to obtain transgenic offspring, which is namely called DMSO-sperm mediated gene transfer (SMGT). Mouse sperm could be either directly transfected via injection into testis or cultured in vitro with the plasmed DNA containing the enhanced green fluorescent protein (EGFP) that could be expressed in the embryos and offspring. Then, 36 living transgenic rabbits were produced using the same technology, and the transgenic ratio of 56.3% was detected using PCR and Southern blot. As the controls, the transgenic ratios of 39.6% and 47.8% have also been tested using the liposomes mediated technology of Tfx-50 Reagent or Lipefectamin-2000, respectively. The results show that the female transgenic rabbits, as the mammary gland bioreactor models, could express the human tissue plasminogen activator mutant (htPAm) in their mammary cells when they are adult.
Available from: Bjorn Oback
- "Several methods were tested to improve uptake of DNA into sperm. These include electroporation (Gagne et al. 1991, Rieth et al. 2000), lipofection (Bachiller et al. 1991, Hoelker et al. 2007) and magnetofection (Kim et al. 2010) as well as addition of DMSO (Kuznetsov & Kuznetsova 1995, Li et al. 2006, Shen et al. 2006) or protamine (Alderson et al. 2006). Irrespective of the transfection method, the mode of transferring exogenous DNA from the sperm into the oocyte via fertilisation is not well understood. "
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ABSTRACT: Transgenic mammals have been produced by using sperm as vector for exogenous DNA (sperm-mediated gene transfer or SMGT) in combination with artificial insemination (AI). Our study evaluated if SMGT could also be achieved in combination with in vitro fertilisation (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, whilst 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 GFP-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP-positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP-fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, intracytoplasmic sperm injection 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.
Reproduction 11/2012; 145(1). DOI:10.1530/REP-12-0340 · 3.17 Impact Factor
Available from: Massimo De Felici
- "Denuded oocytes were divided into four groups: oocytes alone (denuded oocytes, DOs), oocytes alone with 100 ng/ml ActA  (DOs+ActA), oocytes cocultured onto PAGCs (DOs+PAGCs) and oocytes cocultured onto PAGCs in the presence of ActA (DOs+ PAGCs+ ActA). All cultures were carried in drops of 100 µl of medium in 6 cm dishes covered with 6 ml of mineral oil. "
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ABSTRACT: In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day-old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
PLoS ONE 07/2012; 7(7):e41771. DOI:10.1371/journal.pone.0041771 · 3.23 Impact Factor
Available from: Paulo Varoni Cavalcanti
- "These results are consistent with those obtained with rabbits (Kuznetsov et al. 2000): sperm cell permeability to DNA increased from 28% to 81%, but the factors involved in this high success rate were not elucidated. More recently, Shen et al. (2006) and Li et al. (2006) demonstrated the efficient production of transgenic rabbits and mice by TMGT using DMSO-treated DNA. Although the DMSO reduced sperm motility, fertilization and hatching rates were not affected (Li et al. 2006). "
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ABSTRACT: Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.
Journal of Biosciences 09/2011; 36(4):613-20. DOI:10.1007/s12038-011-9098-x · 2.06 Impact Factor
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