Effect of Optimized Treatment of Donor Cells on the Efficiency of Production of SCNT-Cloned

Sun Woo Park(1,2), Yeon Woo Jeong(1), loong loo Kim(1), Kyeong Hee Ko(1) ,Se Heon Jeong(1,2),Yeon IK Jeong(1),Hye Young Son(1),Mohammand Shammim Hossein(1),Yeun Wook Kim,Sang Hwan Hyun(1,2),Taeyoung Shine and Woo Suk Hwang SooAm Biotech Research Foundation, 1024-39 Saam-ri, Wonsam-myeon, Cheoin-gu, Yongin-si, Gyeonggi-do, 449-872,south Korea;Laboratory of Veterinary Biotechnology, College of Veterinary Medicine,Chungbuk National University, 410 Sungbong-ro, Heungduk-gu, Cheongju, 361-763 South Korea Correspondence Author. hwangws@sooam.org; shhyun(@cbuac.kr

ABSTRACT

Somatic cell nuclear transfer (SCNT) is an alternative potential mol for the conservation of endangered. In this study, somatic cells were collected from a purebred 9-month-old male mastiff and an I1-month-old female mastiff. Oocytes that had been matured in vito were retrieved from outbred dogs by laparoscopy. We
used cycling cells as donor cells for SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then fused/activated simultaneously by electrical stimulation. Finally, 224 embryos were transferred to 16 recipients that had been synchronized naturally. The efficiency of delivery of cloned dogs (7.1%) was threefold higher than in previous reports. Moreover, one surrogate delivered four identical cloned female Tibetan Mastiff puppies; another three surrogates each delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. Thus, our study has demonstrated techniques that improve significantly the overall efficiency of SCNT in the canine species.

Cheminls: All chemicals were p echased from Sigma.Aldrich Corp. (St. Louis, MO), unless otherwise stated.

Estimation of time of mutation: The time of ovulation was estimated from the serum progesterone concentration,as reported by Hase et al. (2000). The day on which the progesterone concentration initially reached 40 ng/ml or more was regarded as the day of ovulation. On vaginal cytology during ovulation, superficial epithelial cells usually represented greater than or equal to 90% of the epithelial cells (Evans and Savage, 1970).

Collection of oocvtes, preparation of donor cells and SCNT: All surgeries were performed under general anaesthesia using the procedure published by Kim et al. (2009). Dorm nuclei were obtained from canine fibroblasts using skin biopsies taken from a purebred 9-month-old male mastiff and an 11-month-old female mastiff. Prior to SCNT, cells were thawed and cultured for 3-4 days until confluent- Individual cells were retrieved from monolayers by trypsiniretion for 30 s. The SCNT procedure was modified from that reported by Kim etal. (2009).

Determination of cell cycle and cell death by FACS:
The cellular DNA content was determined by staining cells with propidium iodide and measurement of fluorescence in a Becton Dickinson FACScan (FACSCalibur, BD; San Jose, CA). Canine fibroblasts were harvested by trypsinization and fixed in cooled 70% methanol (-200C). The cells were then incubated for 2 h in a solution containing I mg/ml RNase and 20 pg/ml propidium iodide. Subsequently, the cells were analysed in a Becton Dickinson FACScan (FACSCalibur, BD; San Jose, CA). The proportions of cells in the GO/G I, S, and G2/M phases and those undergoing apoptosis were estimated using the Modfit cell cycle analysis program (CellQuest, BD).

Cell proliferation assays: The CASFs from early and late passages were cultured in 96-well microtitre plates in DMEM supplemented with 10% FBS. The cell proliferation assay was performed using an EZ-CyTokox kit (Daeillab, FZCyTox, Korea) according to the manufacturer's manual. Briefly, 10 pL of kit reagent (10% of culture medium) was added to each well after 24-96 h of incubation. The cells were incubated for 2 h and then shaken thoroughly for I min on a shaker. The absorbance of the treated and untreated samples was measured using a microtitre plate reader at 450 run,according to the filters available for the microplate reader (Molecular Devices, SpectraMax M2/M2e).

Real-time quantitative PCR analysis: To analyse the relative abundance of mRNA of the P/k-1 /4 genes, CASFs were harvested on day 4 after thawing. Messenger RNA was extracted using the RNeasy mini kit (Qiagen, Hilden,Germany) according to the manufacturers instructions.For reverse transcription, 1.5 pg total RNA per 40 pL reaction mixture was converted to complementary cDNA using an M-MLV reverse transcriptase kit (InvitrogenCorporation, Carlsbad, CA). Real-time PCR amplification was conducted using the AB17300 Real-Time PCR System (Applied Biosystems, Foster City, CA). A Power SYBR Green PCR Master Mix (Applied Biosystems,Foster City, CA) was used to provide real-time PCR quantification of the desired PCR products. The real-time PCR reaction mixture consisted of 50 ng of cDNA and I pI each of forward and reverse primers (see Table 3) in a total volume of 20 pl. Three replications were performed,and the mRNA level of each sample was normalized to GAPDH mRNA levels. The relative levels of mRNA were determined by the ratio of 2 ^AA where Ct - threshold cycle for target amplification, ACt - Cttarget gene (Plk-Ior PIk-4) - ACtintemal reference (GAPDH), and AACt -ACt sample - ACt calibrator.

Embryo transfer. The cloned embryos were transferred surgically into the oviducts of naturally synchronized females after simultaneous fusion and activation- The surgeries were performed using the same procedure published by Kim et al. (2009)

Statistical analysis: Differences among treatments were analysed with SPSS using one-way analysis of variance (ANOVA). Data are expressed as means * SD.Comparisons of mean values among treatments were performed using Tukey's least significant difference(LSD) test. Descriptive statistics are used to present the data. Data were analysed using the unpaired t-test with Welch's correction. In each case, a P value of x.05 was considered significant.

Cell cycle analysis of CASFs: To reduce the detrimental effects of synchronization treatments, contact inhibition and serum starvation, we analysed the cell cycle stages in cycling cells by FACS (Table 1). The G0/G1 rates of cycling at day I (p2dl, 61.1%), day 2 (p2d2, 69.5%) and day 3 (p2d3, 76.1%)were significantly lower than for synchronized cells (P<0.05). No significant differences in the GO/G1 stage of cycling cells at day 4 (p2d4, 88.1%),contact inhibition (85.7%) and senun starvation (93.4%)were observed. The proportion of cells in the S phase wider conditions of serum n starvation (1.6%) was significantly lower than other conditions (P<0.05). This indicates that cells cycling at day 4 are able to induce a natural synchronization of the cell cycle without artificial treatments.

Anal sis of apoptosis and proliferation at different passages: Given the result of experiment 1, cells cycling at day 4 were used subsequently as the optimal condition for natural synchronization. To evaluate the effect of cell passage number on cell proliferation and the rate of apoptosis, we used passages 2, 4, 7, 10 and 1 t of cycling cells (Fig, t; Table 2). As shown in Fig. t, the rate of proliferation of cells at passage II (O.D. 12210.15) was significantly lower than that of cells at passage 2 (0-13-1.671004) (Pc0.05), but it was not different from those at passage 4 (O.D. 1.5610.08), passage 7 (O.D. 150:0.05)or passage 10 (O.D. 1.5110.10). Thus, passage 11 was excluded from the analysis of apoptosis. In the analysis of cell cycle phase and apoptosis at different passages (Table2), we observed that, after passage 7, there was a tendency to decrease the proportion of cells in the GO/GI phase.
The G0/G1 rate of cells at passage 10 (plOd4, 72.6%) was significantly lower than at passages 2 and 4 (P<0-05), but it was not different from passage 7 (Table 2). The rate of apoptosis in cells at passage 10 (16.0%) was higher than at the other passages.

Expression of P1k genes in CASFi: We performed semi.quantitative reverse transcriptase polymerise chain reaction (RT-PCR) and real-time PCR (Table 3) to monitor cell cycle synchronization at the molecular level by measuring the expression of cell cycle-regulated P/k genes at passages 2, 4, 7 and 10. Samples of adult skin fibroblasts cycling at day 4 were tested by RT-PCR amplification. Semi-quantitative multiplex RT-PCR. using the housekeeping GAPDH gene as an internal standard,showed that all passages expressed the P/k-I genes (Fig.2A). Using the P/k-I expression level of passage 2 cells as a standard (control,1.00 arbitrary unit), the expression level of P/k-1 mRNA in passage 4 cells (0.70 arbitrary units) was found to be significantly higher than that in passage 7 (0.57 arbitrary units) or passage 10 (0.32 arbitrary units) (P<0.05). The level of expression of P/k-4 mRNA in passage 4 (0.3 arbitrary units) was also significantly higher than in passages 7 and 10 (Fig. 2B).In passages 7 and 10, only faint bands (measuring 0.10 and 0.05 arbitrary units) of P/k-4 mRNA were detected.Thus, the semi-quantitative multiplex RT-PCR assay of Plk-1/-4 was found to be a reliable tool for monitoring the cell viability of canine fibroblasts.

FLY 2:Real-time quantitactive PCR of PLK-L and PLK-4 gene transcripts .the relative abundance of mRNA

transcripts of the PK-L(A)and PLK-4 (B) genes was messured at different passages (2,4,7and 10)of adult skin

fibroblast cells.Three replications were performs three times and the mRNA level of each sample was

normalized to that of GAPDH mRNA