Cats cloned from fetal and adult somatic cells by nuclear transfer

Abstract

This work was undertaken in order to study the developmental competence of nuclear transfer (NT ) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.

In Vitro Development and Postvitrification Survival of Cloned Feline Embryos Derived from Preadipocytes

“…Constant development (blastocyst formation: 20%) of the SCNT embryos reconstructed with IVM oocytes was achieved regardless of the cell cycle synchronization for donor cells. This is similar to or better than those obtained by other groups using fetal fibroblasts (4.0-33.0%) [10,21,22], cumulus cells (2.6-7.7%) [22,36], epidermis-derived fibroblasts (6.1-8.5%) [10,22] or uterus-derived fibroblasts (20.3-30.0%) [37,38]. While further investigation is needed to determine whether the tissue stem cells or precursor cells can be used in SCNT as advantageous nuclear donor cells, our previous studies have demonstrated that cloned animals can be obtained efficiently by SCNT using porcine preadipocytes or salivary gland-derived progenitor cells as donor cells [23,24,33,34].…”

“…Subsequently, the birth of cloned animals from donor cells without cell cycle synchronization has been reported [3]. Cloned cats have also been produced using both cells that were subjected to cell cycle synchronization [8,10] and cells that were not synchronized [7]. In this study, cell cycle synchronization by serum starvation affected neither the rate of fusion between donor cells and recipient oocytes nor the in vitro development of the SCNT embryos.…”

“…Feline SCNT techniques can be applied to the conservation of endangered wild feline species [8] or the preparation of feline embryonic stem cells (ES cells) [9]. However, the current utility of SCNT in such applications is limited because the production efficiency of cloned cats is low [7,8,[10][11][12]. Thus, it is essential to establish a more efficient system to promote the use of SCNT in cats.…”

“…However, there have been few studies investigating such factors in cats [10,11,21,22], so further research is necessary. We had previously demonstrated that cloned piglets could be efficiently obtained using preadipocytes (derived from mature adipocytes) as donor cells in porcine SCNT [23,24].…”

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

“…In feline species many of the strategies suggested to improve SCNT have not had much effect (Yin et al 2005, Gó mez et al 2011, Imsoonthornruksa et al 2011 The majority of the cloned embryos that have been produced so far have shown deficient nuclear reprogramming leading to failures in development to term (Tamada & Kikyo 2004, Sawai 2009). Fetal abnormalities were reported in African wildcat and sand cat cloned fetuses (Gómez et al 2006(Gómez et al , 2008; these may have been associated with alterations in the expression of several genes and epigenetic disorders in donor cells (Gómez et al 2008).…”