Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro.

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs.

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells.

The present study was carried out to examine the effects of post-activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo-β-galactosidase C gene (removal of the α-galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.

Cre-loxP system as a versatile tool for conferring increased levels of tissue-specific gene expression from a weak promoter.

Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo.

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively.

Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C.

The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence.

Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells.

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002).

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay.

A 210-kb segment of tandem repeats and retroelements located between imprinted subdomains of mouse distal chromosome 7.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes.

Structural and functional analysis of a 0.5-Mb chicken region orthologous to the imprinted mammalian Ascl2/Mash2-Igf2-H19 region.

Previous studies revealed that Igf2 and Mpr/Igf2r are imprinted in eutherian mammals and marsupials but not in monotremes or birds. Igf2 lies in a large imprinted cluster in eutherians, and its imprinting is regulated by long-range mechanisms. As a step to understand how the imprinted cluster evolved, we have determined a 490-kb chicken sequence containing the orthologs of mammalian Ascl2/Mash2, Ins2 and Igf2.