Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

Background Cardiac catheterization remains the standard diagnostic technique for assessing both anatomy and physiology in congenital heart disease (Chd). fixed projection angiography (fpa) is the mainstay for guiding congenital cardiovascular interventions. however, fpa has limitations in soft tissue visualization and precise characterization of complex structures such as segmental branch pulmonary arteries, coronary arteries, and anomalous or stenotic pulmonary veins. these limitations are due in part to simultaneous opacification of overlying structures, foreshortening of structures if the projection is not perfectly aligned, and the inability to visualize structures without injection of contrast. integration of 3-dimensional (3d) image data sets with fluoroscopy can potentially overcome limitations of 2-dimensional (2d) angiography for visualizing complex vascular structures and can facilitate accurate diagnoses as well as guide interventional procedures. the use of 3d images obtained from 3d rotational angiography (3dra), Ct, and Mri was originally developed for neuroradiographic endovascular procedures 1-3 but has played an increasing role in cardiovascular medicine for anatomic delineation 4-7 and electroanatomic mapping. 8 rotational angiography (ra) and 3dra have emerged as promising modalities applicable to congenital cardiac diagnosis. 7, 9 additionally, integrated 3d images from 3dra, Ct, and Mri overlaid onto live fluoroscopy are now being used for roadmaps to guide Chd diagnostic and interventional procedures. 5, 6, 10 the potential benefits of integrating 3d images into fluoroscopic procedures for Chd are many, including: (1) improved diagnostic and interventional efficacy, (2) reduced overall radiation exposure, (3) reduced contrast dose, and (4) reduced procedural time. 11-13 all of these are particularly advantageous in the pediatric population. parallel to this, 3d transesophageal echocardiography (3d-tee) is currently being applied to Chd catheterization procedures. this technology allows real-time anatomic visualization of soft tissue structures and catheter guidance within the beating heart.

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