Abstract
The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-Nl vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for 30 μsec. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.
Highlights
A number of methods have been developed to introduce genetic material into the animal genome
Sci. 21(3):358-363 (Perry et al, 1999). Since such procedure does not require zygote with visible pronuclei, it may be a practical alternative to the pronuclear microinjection in the pig because the pronucleus injection in this species is often difficult due to the opaque ooplasm
Porcine sperm heads pre-incubated with green fluorescent protein (GFP) gene construct were injected into oocytes, and without compromising viability of the cells an expression of GFP based on emission of fluorescence was used as an indicator of transgenesis
Summary
A number of methods have been developed to introduce genetic material into the animal genome. Direct microinjection of recombinant DNA into a pronucleus of a zygote (Gordon et al, 1980) has been commonly used to produce transgenic animal and, especially in mice, provided powerful tool for the study on the regulation of gene. In an attempt to improve efficiency of transgenic animal production, the intracytoplasmic sperm injection (ICSI) technique has been combined with sperm-mediated gene transfer to produce transgenic mice with high efficiency. The production efficiencies of transgenic pigs from ICSI embryos have been disappointingly low, for instance, 1 transgenic piglet from 390 embryos transferred (Kurome et al, 2006) and 1 transgenic piglet from 452 embryos transferred (Yong et al, 2006), suggesting that this new procedure still needs to be improved. The present study was to refine the ICSI procedure to facilitate the transgenesis in porcine embryos and to improve the efficiency of producing transgenic pigs. Porcine sperm heads pre-incubated with green fluorescent protein (GFP) gene construct were injected into oocytes, and without compromising viability of the cells an expression of GFP based on emission of fluorescence was used as an indicator of transgenesis
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