Production Efficiency and Telomere Length of the Cloned Pigs Following Serial Somatic Cell Nuclear Transfer

Abstract

Abstract. The aim of the present study was to examine the production efficiency of cloned pigs by serial somatic cell nuclear transfer (SCNT) and to ascertain any changes in the telomere lengths of multiple generations of pigs. Using fetal fibroblasts as the starting nuclear donor cells, porcine salivary gland progenitor cells were collected from the resultant first-generation cloned pigs to successively produce second-and third-generation clones, with no significant differences in production efficiency, which ranged from 1.4% (2/140) to 3.3% (13/391) among the 3 generations. The average telomere lengths (terminal restriction fragment values) for the first, second and third generation clones were 16.3, 18.1 and 20.5 kb, respectively, and were comparable to those in age-matched controls. These findings suggest that thirdgeneration cloned pigs can be produced by serial somatic cell cloning without compromising production efficiency and that the telomere lengths of cloned pigs from the first to third generations are normal. Key words: Aging, Pig cloning, Progenitor cell, Somatic cell nuclear transfer (SCNT), Telomere (J. Reprod. Dev. 54: [254][255][256][257][258] 2008) ince the birth of the first cloned sheep, more than 10 animal species have been cloned by SCNT [1]. However, various questions and problems remain with regard to production of somatic cell clones and practical application of cloning techniques. For example, abnormal development of cloned fetuses, anatomical abnormality of cloned offspring, postpartum mortality and low production efficiency of healthy clones are problems commonly encountered with all animal species [2][3][4].Somatic cell cloning comprises replication of an individual animal. It is of considerable biological significance to ascertain the capability of SCNT technology in producing multiple generations of clones, namely, sequential cloning of cloned animals. To date, mice have been cloned up to six generations [5], while cows have been cloned up to two generations [6].Serial cloning of pigs has practical implications in producing genetically engineered animals. Genetically modified pigs such as alpha 1,3-galactosyltransferase-knockout pigs [7][8][9][10][11][12][13][14] and those with a n-3 fatty acid desaturase gene [15] have been produced using serial cloning technology. In this context, multiple generations of clones up to the third generation have been reported in pigs [16,17]. On the other hand, studies have found that serial cloning causes the accumulation of epigenetic modifications that result from the SCNT procedure [3,18]. Indeed, the production efficiency of clones has been reported to decrease as the number of serial clonings increases [5,6,17].Telomere shortening is another form of genetic modification resulting from the somatic cell cloning procedure [19][20][21][22][23][24]. However, serial somatic cell cloning has been shown to not cause telomere shortening in mice [5] and cows [6]. The changes in telomere length involved in serial pig cloning have yet to be determined....

Cloning of Homozygous a1,3-Galactosyltransferase Gene Knock-Out Pigs by Somatic Cell Nuclear Transfer

“…With multiple gene modifications in pigs, the feasibility of serial somatic cell cloning (multigenerational cloning) would have important implications. In this regard, we have successfully produced the 4th generation of cloned pigs by serial cloning (Kurome et al, 2008a;Matsunari et al, 2008a). We have developed a process for multi-generational cloning of pigs, that is, a means of proceeding from the first clone generation (G1) to the second (G2) and then to the third generation (G3), and the efficiency of producing cloned pigs throughout this process was almost constant (Kurome et al, 2008a;Matsunari et al, 2008a).…”

“…In this regard, we have successfully produced the 4th generation of cloned pigs by serial cloning (Kurome et al, 2008a;Matsunari et al, 2008a). We have developed a process for multi-generational cloning of pigs, that is, a means of proceeding from the first clone generation (G1) to the second (G2) and then to the third generation (G3), and the efficiency of producing cloned pigs throughout this process was almost constant (Kurome et al, 2008a;Matsunari et al, 2008a). Notably, the cloned offspring did not show any shortening of their telomeres (Kurome et al, 2008a) and grew normally.…”

“…We have developed a process for multi-generational cloning of pigs, that is, a means of proceeding from the first clone generation (G1) to the second (G2) and then to the third generation (G3), and the efficiency of producing cloned pigs throughout this process was almost constant (Kurome et al, 2008a;Matsunari et al, 2008a). Notably, the cloned offspring did not show any shortening of their telomeres (Kurome et al, 2008a) and grew normally. Our results indicate that serial cloning is a feasible option for production of specifically designed pigs with multiple genetic modifications.…”

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

“…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]. Because the fat tissues where preadipocytes are found are near the body surface, these cells can be collected in a less invasive manner compared with stem cells derived from bone marrow and viscera [23,24,30].…”

“…Nuclear transfer of feline preadipocytes was performed according to the method used previously for porcine SCNT, with slight modification [23,[32][33][34]. Oocytes matured in vivo or in vitro were treated with 1 mg/ml hyaluronidase and denuded of cumulus cells by pipetting.…”