48 THE AMOUNT OF TELOMERIC DNA IN LYMPHOCYTES OF DOG CLONES

Controversy regarding the restoration of eroded telomere length of donor cells after the nuclear transfer process has arisen from previous studies of cloned cattle, mice, and pigs. Little is known about telomere lengths in dogs from either natural breeding or somatic cell nuclear transfer (SCNT). In this study, we investigated the amount of telomeric DNA (ATD) in the lymphocytes of growing dog clones and their somatic cell donors. One cloned male Afghan hound dog [Snuppy (Lee et al. 2005 Nature 436, 641)] and 3 cloned female Afghan hound dogs (Jang et al. 2006 Theriogenology; doi:10.1016J.THERIOGENOLOGY.2006.11.006) were obtained from somatic cell nuclear transfer (SCNT) of ear skin fibroblasts. The lymphocytes were recovered from all dog clones: their nuclear donor dogs (male donor dog, female donor dog), and six normal Afghan hound dogs (control, and 10-, 20-, 26-, 49-, 55-, and 58-month-old, respectively). The ATD was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a telomeric DNA probe. A telomeric probe containing the TTAGGG repeated DNA sequence was simultaneously amplified and labeled with digoxigenin (DIG) by polymerase chain reaction (PCR) using dog genomic DNA as template, a (GGGTAA)7 primer, and a DIG-labeling kit (Roche, Mannheim, Germany). To analyze the amount of telomeric DNA on the lymphocytes, at least 100 interphase nuclei were examined in each specimen. The image was captured by a digital camera (DP-70, Olympus) and analyzed using MetaMorph (Universal Imaging Co., Downingtown, PA, USA), an image analysis program. Our results indicated that the ATD in normal Afghan hounds gradually decreased with age. Although no difference in ATD was observed between 10- and 26-month-old dogs, the ATD in the 26-month-old dog was significantly higher than that in 49-, 55-, and 58-month-olds (P < 0.05). The mean percentage of telomeric DNA in Snuppy (18-month-old; 2.38%) was significantly higher than that in the nuclear donor dog (49-month-old; 2.12%) but less than that in the age-matched control (20-month-old; 2.72%; P < 0.05). The ATD in 3 female clone dogs (3-, 2-, and 2-month-olds; 3.47, 3.28, and 3.07%) were significantly higher than that in the nuclear donor (26-month-old; 2.65%). In conclusion, the mean percentages of telomeric DNA in dog clones were higher than in nuclear donor dogs, and the ATD of the cloned male dog was different from that in age-matched controls from natural reproduction. The results suggest that the amount of telomeric DNA in dog clones can be restored with the nuclear transfer of cultured donor fibroblasts, but further studies are required as to how telomere reprogramming occurs during the nuclear transfer process.

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24 DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF EMBRYONIC CULTURED CELLS ISOLATED FROM SOMATIC CELL NUCLEAR TRANSFER BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab24 ◽

2008 ◽

Vol 20 (1) ◽

pp. 92

Author(s):

X. J. Bai ◽

J. L. Yu ◽

M. Murakami ◽

Y. Zhang ◽

Y. J. Dong

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cultured Cells ◽

Es Cells ◽

Donor Cells ◽

The Mean ◽

Development In Vitro ◽

Chi Square Analysis

Embryonic stem (ES) cells derived from somatic cell nuclear transfer (NT) bovine embryos would increase the utility of the cow as a large animal model for human cell therapy. They would also be useful for studies of cell differentiation. Such cells exhibit full pluripotency, and cloned offspring were obtained from them following a second NT in mice, indicating that the reprogramming that produced pluripotent ES cells could be reversed (Wakayama et al. 2001 Science 292, 740–743). The objective of this study was to examine if there would be any beneficial effects of using somatic cell NT-derived embryonic cultured cells as donors for cloning in cattle. Cloned embryos were produced from a single cell line of bovine fetal fibroblasts (FF) and adult ear-tip cells (AEC) (passages 1 to 5) by NT, as described previously (Dong et al. 2004 Asian–Aust. J. Anim. Sci. 17, 168–173). NT embryos that reached the blastocyst stage were cultured separately to isolate embryonic cultured cells derived from FF (NT-FF) and AEC (NT-AEC) according to previous methods (Dong et al. 2003 Acta Genet. Sin. 30, 114–118). More than 80% of the generated embryonic cultured cells stained positive for alkaline phosphatase. Embryonic cells cultured for 7 to 35 days were used as the donor cells for NT in the NT-FF and NT-AEC groups. Cloned embryos were produced using individual cell lines of FF, AEC, NT-FF, and NT-AEC (passages 1 to 5, putative cell cycle stage of G0 or G1) as donor cells, and their development in vitro is summarized in Table 1. The FF and AEC groups include data from the initial round of NT. The rates of fusion and embryo development were compared by chi-square analysis. Duncan's multiple range test was used to compare the mean cell numbers of blastocysts. The percentage of embryos that developed into blastocysts was significantly higher (P < 0.05) in the FF group than in the AEC group. Interestingly, we observed that the developmental potential in vitro and the mean cell number of blastocysts tended to be higher in the NT-FF and NT-AEC groups than in the FF and AEC groups. A total of 15 and 6 good quality Day 7 embryos in the NT-FF and NT-AEC groups were nonsurgically transferred to 5 and 3 synchronized recipients (2 to 3 embryos/female), respectively. On Day 30 of gestation, 3 (60%) and 1 (33%) females in the NT-FF and NT-AEC groups, respectively, were diagnosed as pregnant via ultrasonography. One (20%) recipient cow in the NT-FF group remained pregnant at Day 60 of gestation, but lost the pregnancy by Day 90. These results suggest that cloning of bovine embryonic cultured cells generated from fetal and adult somatic cells by NT can produce transferable embryos and initiate pregnancies, although none of the pregnancies has developed beyond the first trimester at this time. Table 1. Development in vitro of bovine NT embryos produced from different donor cell types

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356 IMPROVED ENUCLEATION EFFICIENCY FOR PIG SOMATIC CELL NUCLEAR TRANSFER BY DENUDING OOCYTES AT 30 HOURS OF IN VITRO MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab356 ◽

2007 ◽

Vol 19 (1) ◽

pp. 293 ◽

Cited By ~ 1

Author(s):

K. Song ◽

J. Park ◽

E. Lee

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Polar Body ◽

Digital Camera ◽

Cumulus Cells ◽

Cell Number ◽

Blastocyst Formation ◽

Significant Difference

Oocytes for somatic cell nuclear transfer (SCNT) have to be removed from their cumulus cells before enucleation. Denuding oocytes by vortexing or repeated pipetting makes the polar body (PB) deviate from the metaphase (MII) plate, which in turn makes it difficult to remove DNA materials completely during enucleation. We hypothesized that denuding oocytes at 30 h of IVM maintains the MII plate and PB in a closer position and therefore makes it easy to enucleate. To test this hypothesis, oocytes were matured in TCM-199 supplemented follicular fluid, hormones, EGF, cysteine, and insulin for first 22 h, and in a hormone-free medium for 18 h with three modifications: (1) cumulus cells were removed from oocytes just prior to enucleation at 40 h of IVM (control), (2) oocytes were denuded at 30 h of IVM and co-cultured with their detached cumulus cells for 10 h (D+), and (3) oocytes denuded at 30 h of IVM were cultured without cumulus cells (D-). After IVM, some oocytes were stained with Hoechst 33342 and photographed by a digital camera; the distance between the MII plate and the PB were measured using an image analysis program (ImageJ 1.36; http://rsb.info.nih.gov/ij). Also, the enucleation rate after blind enucleation and the in vitro development of SCNT embryos were determined. For SCNT, oocytes were enucleated, and nuclear material from donor cells (skin fibroblasts from a miniature pig) was inserted; oocytes were then electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park et al. 2005 Zygote 13, 269-275) for 6 days. Embryos were examined for their cleavage and blastocyst formation on Days 2 and 6, respectively (the day of SCNT was designated Day 0). Data were analyzed by the GLM procedure and the least significant difference test in SAS (SAS Institute, Cary, NC, USA). The distance between the MII plate and the PB was significantly (P < 0.01) shorter in D+ and D- embryos (19.4 and 18.9 �m, respectively) than in the controls (25.5 �m). Enucleation rates after blind enucleation were significantly (P < 0.01) higher in D+ and D- groups (77% and 72%, respectively) than in the controls (60%). Oocyte maturation (89–91%), SCNT embryo cleavage (71–77%), blastocyst formation (4–5%), and embryo cell number (39-45 cells/embryo) were not altered by different denuding methods. The perivitelline space (PVS) increases with time during maturation and denudation, after PB extrusion markedly enhances PB deviation. It is likely that increased PVS in control oocytes enhanced PB deviation during denudation and then resulted in lower enucleation rate. In conclusion, the results of this study indicated that denuding at 30 h of IVM maintained the MII plate and the PB in a closer position and improved enucleation efficiency without impairing developmental capacity of SCNT embryos. This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

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Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise

Reproduction ◽

10.1530/rep.1.01203 ◽

2007 ◽

Vol 133 (1) ◽

pp. 243-255 ◽

Cited By ~ 40

Author(s):

C J Fletcher ◽

C T Roberts ◽

K M Hartwich ◽

S K Walker ◽

I C McMillen

Keyword(s):

Somatic Cell ◽

Gestational Age ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Caspase 3 ◽

Fetal Death ◽

Fetal Demise ◽

Structural Abnormalities ◽

The Mean ◽

And Function

The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with ~5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT diein utero. Placentomes from SCNT pregnancies (n= 9) and age matched, naturally mated controls (n= 20) were collected at two gestational age ranges (105–134 days and 135–154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105–134 days: control 55.0 ± 4.2, clone 44.7 ± 8.0 and 135–154 days: control 72.2 ± 5.1, clone 36.6 ± 5.1;P< 0.001) and increased the mean individual placentome weight (105–134 days: control 10.6 ± 1.3 g, clone 18.6 ± 2.8 g and 135–154 days: control 6.6 ± 0.6 g, clone 7.0 ± 2.0 g;P< 0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P< 0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently, the volume of intact trophoblast was reduced and the arithmetic mean barrier thickness of trophoblast through which exchange occurs was altered (P< 0.001) at both gestational age ranges in clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones.

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