Production of cloned pigs with targeted attenuation of gene expression.

The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

Pre- and postimplantation development of swine-cloned embryos derived from fibroblasts and bone marrow cells after inhibition of histone deacetylases.

The present study assessed changes in epigenetic markers and pre- and postimplantation development in somatic cell nuclear transfer (SCNT) porcine embryos after treatment with the inhibitor of histone deacetylases (HDACi), Trichostatin A (TSA). Embryos were generated using in vitro matured oocytes and nuclei from either a male fetal fibroblast (FF) cell line or bone marrow cells (BMC) from three adult sows. After nuclear transfer, oocytes were either exposed or not to 10 ng/mL TSA for 10 h starting 1 h after cell fusion. Samples of one-cell stage and cleaved (two- to four-cell stage) embryos were fixed at 15 to 18 h or 46 to 48 h after cell fusion and immunocytochemically processed to detect histone H3 acetylation at lysine 14 (H3K14ac) or histone H3 dimethylation at lysine 9 (H3K9m2) using specific primary antibodies. TSA treatment increased the immunofluorescent signal for H3K14ac in cleaved embryos derived from both FF and BMC but did not affect H3K9m2. Development to the blastocyst stage was increased by TSA treatment (45.2 vs. 23.9%) in embryos produced from FF cells but not in those produced from BMC (30.6 vs. 27.4%). Cloned piglets were produced from both treatments when day 5 to 6 blastocyst-stage embryos derived from FF cells were transferred into the uterus of recipient females. Cloned piglets were also produced after the transfer of TSA-treated blastocysts derived from BMC of adult sows but not from control embryos. These findings suggest that the inhibition of histone deacetylases have similar effects on enhancing H3K14ac in SCNT embryos reconstructed from different cell types but the effect on in vitro and in vivo development seems to differ according to the nuclear donor cell type.