Article

Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method.

Department of Life Science, Peking University, Beijing, China.
Molecular Reproduction and Development (Impact Factor: 2.81). 06/2006; 73(5):589-94. DOI: 10.1002/mrd.20401
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
38 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian spermatozoa is a unique cell in many ways and the acquisition of its main function, i.e. the fertilization capacity is a multi-step process starting in the male gonad and ending near the female egg for the few cells reaching this point. Due to the unique character of this cell, the molecular pathways necessary to achieve its maturation also show some specific characteristics. One of the most striking specificities of the spermatozoa is that its DNA is highly compacted after the replacement of histones by protamines, making impossible the classical processes of transcription and translation. The sperm cells are thus totally dependent on their extracellular environment for their protection against oxidative stress for example, or for the molecular changes occurring during the epididymal transit, the first organ in which the post-testicular maturation takes place. The molecular mechanisms underlying the sperm maturation are still largely unknown, but it has been shown in the past three decades that extracellular vesicles secreted by the male reproductive tract are involved in this process. This review will examine the roles played by two naturally occurring extracellular vesicles called epididymosomes and prostasomes, respectively secreted by the epididymis and the prostate. We will also present how the use of artificial vesicles, liposomes, contributed to the study of the male reproductive physiology.
    Reproduction 04/2013; · 3.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sperm-mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still underoing development and optimization, to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real-time PCR-based assay to assess the absolute number of exogenous plasmids internalized into the spermatozoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found DNA internalization into the nucleus of porcine sperm cells is better under specific methyl-β-cyclodextrin (MCD)-treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 10/2012; · 2.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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; · 3.56 Impact Factor