Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

Abstract

The sperm-mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore-labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.

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

“…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 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.…”

“…A number of studies have employed indirect methods to investigate this phenomenon. These include liquid scintillation counting of radioactively labelled DNA co-precipitating with sperm (Lavitrano et al 2002, Kang et al 2008, Kim et al 2010, flow cytometry of sperm incubated with fluorescently labelled plasmids (Canovas et al 2010) and genomic PCR assays after incubating sperm with DNase to destroy any non-internalised exogenous DNA (Hoelker et al 2007, Kang et al 2008. While these assays demonstrated association between exogenous DNA and sperm, they provide no direct evidence for DNA uptake across the plasma membrane into the sperm cytoplasm or nucleus.…”

“…While these assays demonstrated association between exogenous DNA and sperm, they provide no direct evidence for DNA uptake across the plasma membrane into the sperm cytoplasm or nucleus. Few studies have addressed DNA uptake into sperm using direct visualisation, such as autoradiography (Brackett et al 1971, Atkinson et al 1991, wide-field (Anzar & Buhr 2006) and confocal laser scanning epifluorescence microscopy of fluorescently labelled sperm (Bachiller et al 1991, Chan et al 2000 or transmission electron microscopy on thin sections (Kim et al 2010). These studies have shown DNA binding to the sperm surface, preferentially in the postacrosomal region, but not provided conclusive evidence for its uptake into sperm with an intact plasma membrane.…”

“…Thirdly, detection of exogenous DNA in embryos after SMGT remains controversial. Many studies have relied on indirect genomic PCR assays that may demonstrate the presence of an exogenous DNA sequence in the embryo or surrounding culture medium but not cellular uptake of this sequence (Rieth et al 2000, Kim et al 2010. Cellular expression of different reporter proteins at various embryonic stages has been analysed.…”

The Use of Nanostructures for DNA Transfection

“…The main goal on use of nanostructures for DNA/RNA transfection is that nanocomposites such as nanotubes, nanoparticles or cationic dendrimers can protect DNA strands against nucleases during cellular delivery acting as a physical barrier to DNase enzymes [28].…”