Handmade Somatic Cell Cloning in Cattle: Analysis of Factors Contributing to High Efficiency In Vitro1

Abstract

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

“…light with a blunt enucleation pipette. Each cytoplast was then individually washed for few seconds in 300 mg/ml phytohemagglutinin P in PBS and quickly dropped over a single donor cell (Vajta et al 2003) settled at the bottom of a microdrop of donor cell suspension diluted in SOF-HEPES. Cell couplets were washed in 0.3 M mannitol (Ca 2C -free, 100 mM Mg 2C ) solution, fused by double DC pulses of 1.2 kV/cm applied for 30 ms, and returned to maturation medium.…”

Genetic Engineering and Cloning: Focus on Animal Biotechnology

“…Another enucleation method is the oocyte bisection [49]. In this case the PZ is removed and the oocytes are sectioned by a micro-blade in two halves, being removed 50% of the cytoplasm.…”

Circles around the farm animal embryo a Danish perspective

“…We have been working on this complex technology over more than 20 years, first in cattle with birth of calves as a result (Smith et al, 1994), since in cattle and pigs with a zona-free approach (Booth et al, 2001a, b), and then with Gabor Vajta's handmade-cloning system (HMC; Vajta et al, 2003;Kragh et al, 2004) that resulted in the first piglets born in 2006 (review by Vajta and Callesen, 2012). The HMC system was going through a number of optimizations for example with different cytoplasmic volumes and with gilt versus sow oocytes Pedersen et al, 2015).…”