Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.fals

Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well

Primary Transgenic Bovine Cells and Their Rejuvenated Cloned Equivalents Show Transgene-Specific Epigenetic Differences

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences

A micro-device integrating vertical and bottom electrodes for 3D cell rotation

We report a Lab-on-chip micro device for rotating a spherical object such as bovine cell in three dimensional (3D) spaces. Micro device consists of two pairs of orthogonal vertical wall electrode along with bottom transparent indium tin oxide (ITO) electrodes. Photolithography technique is employed to fabricate bottom part of the micro device. On the other hand micro milling is applied to fabricate the top electrodes of 500 μm height. The entire device is only 75X25 mm in length and width upon which particle rotation is achieved. Micro device works entirely on this dielectrophoresis (DEP) phenomenon to rotate the cell in 3D spaces. Rotating fields are optimized through simulation and results are presented along with analysis. The experiments are carried out to determine the cell rotation rate. Rotation of cell in 3D spaces has its unique advantages in automation of high throughput mammalian cloning procedures. Cloning procedures such as enucleation requires a cell to be rotated in 3D spaces for genetic material extraction. Enucleation also requires the cell to be precisely rotated and controlled. This precise rotation control is achieved by electrorotation through DEP

Dielectrophoresis Based 3D Cell Rotation through Integration Of Bottom And Vertical Electrodes

Dielectrophoresis rotation of particles in 3D has important applications in biological cell manipulation, sorting and characterization. This paper reports the rotation of spherical bovine oocytes in yaw and pitch axes and their angular velocity spectrum by generating dielectric torque on the oocytes inside the chamber of a fabricated biochip. We predict by numerical computation and confirm experimentally that bovine oocytes are permanently polarizable with no Maxwell Wagner interfacial relaxation at critical frequency. The capability of rotating particles in 3D opens new research opportunities in cell manipulation and analysi

Simulation study of rotational electric field on spherical partical for electrorotation studies

“© 2013 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.”We report three dimensional (3D) rotational (ROT) electric field simulation generated by a biochip to levitate and rotate micron sized bovine cell or a neutrally charged spherical particle. Main goal of this research is to obtain controlled object rotation in yaw, pitch and roll axes for the purpose of automation of enucleation process during bovine cloning. Finite element analysis (FEA) results of rotating electric fields are analyzed for the determination of DEP torque on the cell. This also serves as a necessary tool to determine the dielectric spectrum properties of cells for biologists