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

日期:2017年5月5日 16:11
The efficiency of somatic cell cloning mastiff donor cells has been further optimized
 

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

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ABSTRACT

ARTICLE HISTORY

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.

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Reveived: October 19, 2011
Revised: Docember 01, 011
Acceptd: January 15, 2012
Keywords:
Cloned mastitis
Cycling adult skin
fibroblasts
Plk gene
SCNT
©2012 PVJ. All rights reserved
 
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To Cite This Article: Park SW, YW Jeong, JJ Kim, KH Ko, SH Jeong, Yl Jeong, HY Son, MS Hossein, YW Kim,SH Hyun, T Shin and W S Hwang, 2012. Effect of optimized treatment of donor cells on the efficiency of production of SCNT-coned mastiffs Pak Vet 1, 32(4): 494-498.
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INTRODUCTION

We previously reported successful production of cloned canids, but the efficiency was extremely low (Leeet al, 2005). It is particularly difficult to analyse the in vitro developmental viability of cloned canine embryos because no in vitro culture (IVC) system has been established for them. Although various efforts have been made to establish the technology, the efficiency of the procedure has not improved greatly (Hossein et a!, 2008; Kim et al., 2009). Many factors can affect the efficiency of somatic cell nuclear transfer (SCNT), some of which have been reported to be successful in the development of canine SCNT embryos. These factors include the collection of oocytes in vim as recipient cytoplasm (Hossein et al., 2008), preparation of somatic cells for donor nuclei, the SCNT procedure use(Kimetal.,2009),fusion/activation (Jang er al., 2008; Kim et al.,2009), and embryo transfer (ET) (Fayrer-Hosken, 2007).If these factors were optimized, dogs could be cloned by SCNT with high efficiency.
Polo-like kinases (Plks) are one of the genes involved in cell cycle regulation (Tsvetkov, 2004). Kites et al.(2000) reported that expression of Plks was a suitable marker for determining cell cycle stage in porcine fetal fibroblasts and should lead to the identification of optimal cell culture conditions for donor cells in porcine SCNT.However, expression of Plks in canine fibroblasts had not been reported previously. When non-green fluorescent protein (GFP) transfected cells are used as donor nuclei is SCNT, there is no way to confum their cell cycle stage or viability (Hyun et al., 2003), yet SCNT efficiencydepends on the donor cell cycle stage (Wilmut er al.,1997). However, in studies of SCNT cloned dogs, donor nuclei, which are considered to be the most important factor, have been neglected.Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids, we analysed the effect of cell-cycle synchronisation methods, including the use of cycling canine adult skin fibroblasts (CASFs), or the cell-cycle stage and viability of donor nuclei We then investigated proliferation and P/k-1 /-4 gene expression in CASFs to identify the optimal state of SCNT donor cells.Additionally, we report here an advanced fusion/ activation method and describe the high efficiency in production of cloned dogs using SCNT of optimised cells into enucleated oocytes
 
MATERIALS AND METHODS
 

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.

 
RESULTS
 

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.

 

Production of cloned Tibetan Mastiffs: To assess the effect of the optimized treatment of donor cells on the efficiency of cloning dogs using SCNT, we transferred 224 cloned embryos into the oviducts of 16 surrogate dams by surgical embryo transfer (ET). Fifty-four cloned embryos were transferred into four surrogates as a control group. In control experiments, the pregnancy rate (25%) and production efficiency of dog cloning (1.8%) were similar to the results of the previous report (pregnanc) rate, 22.2% production efficiency, 2.0Y.). Under the conditions of the donor cells described above, donor cells of passages 4 and 5 cultured at day 4 were used for SCNT.The delivery efficiency of cloned dogs (7.1%) was more than three fold higher than when the previous methods.
 
Fly 1: Proliferaction assay of different passages in adult skin fibroblast cells of Tibetan. cells from early (2,p2 )to late (ll,pll)passages were plated on to % well plates (0.1-1×10^6cells/well).Ezcytox reduction was monitored by optical density (O.D) at 450mm.Control represents the blank (p<0.05).Ten replicates were used .

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

 
Table 3:Primers and sizes of amplification products
 
 
Table 3:Effect of optimised treatment of Tibetan Mastiff skin fibroblast cells on production efficiently of SCNT-cloned puppies
 
 
were employed (Table 4). Funhennore, the cloned puppies exhibited a strong phenotypic resemblance to each other as well as to their respective somatic cell donors.
 

DISCUSSION

 
Khamnurtit et al. (2008) reported the donor cells were synchronized at the GO/GI phase by serum starvation or contact inhibition on cell cycle synchronization in canine fibroblasts. In agreement with that report, we also demonstrated that treatment using serum starvation/contact inhibition induced synchroniza-tion at GO/GI (Table 1). However, while artificial synchronization using serum starvation/contact inhibition is used widely to synchronize donor cells, it often increases the proportion of apoptotic cells (Kues er al.,2000) and causes high embryonic losses after SCNT(Lawrence er al., 2005). For this reason, we tried to use a national condition of cycling cells to use instead of the
GO/GI phase distribution used in artificial synchronization; we found that cells cycling at day 4 were in a state that resembled synchronization, but without the detrimental effects. Thus, cell cycle synchronization of donor cells by artificial synchronization is not required for canine SCNT.
The number of donor cell passages is also an important factor in SCNT. Kubota et al. (2000) reported that up to 15 passages of longtemn cultured skin fibroblasts did not decrease cloning competence in bovine
SCNT. Thus, we examined whether their strategy could be applied to canine SCNT using assays of the rates of proliferation and apoptosis (Fig. I and Table 2). In out canine fibroblast cells, however, cells in passage II showed a decreased rate of proliferation. In addition, the rate of apoptosis in cells at passage 10 was significantly higher than in cells at other passages, and cells from passage 7 had a decreased GO/G1 phase. In agreement with our results, Li et al. (2003) demonstrated that the rate of nuclear remodelling decreased significantly when a large number of passages were used in equine SCNT, and
Mammon et al. (2006) reported that apoptotic cell death increased with senescence in normal human skin fibroblast cells. This phenomenon suggests that long-term culture may induce decreased viability and alter genetic or epigenetic regulation in the donor cells. For this reason.we analyzed the expression of cell cycle-regulated Plk genes (Fig. 2). Because the P/k-I gene is primarily responsible for mitotic functions, and also because its protein levels are high in the S and G2/M phases and low after mitosis (Tsvetkov, 2004), we measured the level of expression of P/k-1 mRNA at different passages (p4, p7and p10), using passage 2 as a standard. We therefore analysed centriole biogenesis induced by P/k-4 in mammalian cells at different passages of donor cells.Cells prior to passage 7 had levels of P/k-11.4 mRNAs comparable to normal healthy cells (Fig. 2). This indicates that selection for high expression of P/k-1/-4 in donor cell candidates may compensate for the deficiency in mitotic modulators in the enucleated ooplasm during SCNT.
Using optimized donor cells, we then prepared Tibetan Mastiff cells cultured at day 4 of passages 4 and 5
and attempted the simultaneous fusion/activation method for SCNT. We reported previously that delayed activation with &DMAP was better than protocols based on electrical activation for the production of SCNT cloned puppies (Kim er al., 2009; Hossein et a!., 2009x; Hosseiner al., 2009b). However, in the present study we found that the simultaneous electrical fusion/activation protocol was simple and efficient and that no further chemical stimulation was required for postactivation (Table 4);Hyun et al. (2003) also reported the successful cloning of piglets by SCNT using electrical stimulation alone.
llte 7 ibetan mastiff rs the oldest breed of dog in the world, and it is at the edge of extinction (Li et a1, 2008).Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level.Despite the fact that most Tibetan Mastiff puppies are bom between December and January (because their oestrous period usually takes place during late autumn),we have demonstrated that this optimized SCNT protocol could overcome the limitation of the seasonal reproductive characteristics of Tibetan Mastiffs (this study was carried out in April) by using oocytes and surrogates from other breeds. Therefore, our results suggest that SCNT represents a means of increasing the number of individuals within a population, and that it provides an efficient strategy for the preservation of genetic material,especially in the conservation of extinction-endangered species that reproduce poorly in captivity. Both male and female animals could be cloned and reintroduced into their natural environments. To date, we have also produced SCNT cloned dogs from breeds of all sizes,from small (beagles. Hossein et al., 2009b), to medium (Golden Retriever, Kim et al., 2009; Missy, Hossein et al.,2009a) and large (Tibetan Mastiff). The SCNT procedure can thus be utilized to generate cloned dogs regardless of breed.
 
Conclusion: The present study has demonstrated that: 1) cell-cycle synchronization of donor cells by senuc starvation/contact inhibition is not required, 2) P/k-1/-4 mRNA can be used to select the donor cells, and 3) electrical stimulation alone is sufficient to activate SCNT embryos for the production of cloned dogs by SCNT. As a result, we were able to use somatic cells collected from both female and maleTibetan Mastiffs to produce IC female and 6 male mastiffs.
 
Acknowledgement: In this study, SHH was supported by a grant from Next-Generation BioGreen 21 program#P100812101201I), Rural Development Administration,Republic of Korea.
 
REFERENCES
 
E.am JM and TJ Savage 1970. The collection of vaginal smears from bitches-Hosken Vet Rec, 87: S96599.
Fayrer-Hosken R,2007. Embryo transfer in the dog and cat Theriogenology 68: 382.385.
Hase M, T Horl, E Kawakami and T Tsutsul, 2000. Plasma LH and progester levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs Vet Med Sci 62:243-248.
Hossein Ms YW Jeong Skim JJ Kim SW park CS Jeong SH Hyum and WS Haang,2008 Protocol for the recovery of in vivi matured canine oocytes based on once daily measurement of serum progesterone Cloning Stem Cells,10:403-408.
Hossein Ms YW Leong SW park JJ Kim E Lee KH KO Hs Kim YW Kim SH Hyum T shine L Hawthorone and WS Hwang 2009a
Cloning missy: obtaining multiple offspring of a specific canine
genotype by somatic cell nuclear transfer.Cloning Stem Cells,||:123-130.
Hossein MS YW Jeong SW Park JJ Kim and WS Hwang,2009b ,Birth of Beagle dogs by somatic cell nuclear transfer Anim Reprod Sci 114:404-414.
 
Pak Vet J, 2012, 32(4): 494-498.
 

Hyut S. G Lee. D Kim H KM S Lee, D Naps Y jn nB, S Kim S YeomS Kany J Han. 6 lee and W Ft- & 2003. Produunn of nuclear transfer-derlved Plglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein Biol Reprod 69:1060.1068 Jang G, H Oh, M Kim Y Fibrianto, M Hossein,H Kim .S hong,J Park and S Katt, 2008 I pee.awa d canine somatic cell nuclear transfer procedure. Theriogenology, 69: 146.154. Khammanit R. S Chantakru Y Kitlyanant and j Salkhum 2008. effect of serum starvation and chemcial inhibitors on cell cycle synehronenwon of canine dermal fibroblasts Theriogenology, 7027.34.

Kim S, SW Park. MS Hosseln, YW pon . L Kim, E Lee, YW Kim SH Hyur T SAN and WS Hwary, 2009. ProdAatlan d cloned dogs by decressing the interval between fusion and activation during somatic cell nuclear transfer.Mol Reprod Dew,76:483-489

Kubota C H Yamakuchi j Todoroki. K Mizoshita N Tabaa, M Barber and X Yang 2000 Six cloned calves produced from adult fibroblast

cells after long-term cilture Proc Natl Acad Sci USA, 97: 990.995.
Kun WA M Ang, JW Carnwant I Medlk and H NAn s 2000. Cel
cycle n roniea on d pn dn. 8k eiecu d serum
deprhadnn and reversible cell nyde V#A* i Bid Reprwd 62:412419.

Lowrence JL FN Schrick, FM Hopkins, MG Welborn MD McCracken,T Sonstegard TJ Wlison and JL Edwards 2005. Fetal and pathologic findings of clones derived from serum-starved versus serum-fed bovine ovarian granulosa cells .reprod Biol 5:171-184.

Lee BC, MK Kkm, G Jang, Hj Oh F Yuda, HJ Kim. MS Hossein, JJ Kim
SK Kang G Schatten and WS Hwag, 2005. Dogs cloned from adult somatic cells Nature, 436: 641.

Li X JL Tremaleda and WR Allen. 2003. Effect of the number of passages of fetal and adult fibroblases on unclear remodelling and first ambryonic division in reconsstructed horse oocytes after nuclear transfer Reproduction ,125:535-542.
Li Q.Z Liu,Y Li X Zhao ,L Dong,Z Pan,Y Sun N Li Xu and Z Xie 2008 Origin and phylogenetic analysis of Tibetan Mastiff based on the mitochondrial DNA sequence J Genet Genomics, 355:355-340.

Mammone T D Gan and R Foyouzi-Youssfi 2006 Apoptotic cell death increases with senescence in normal human dermal filroblast cultures cell Biol Lnt 30:903-909
Tsvetkov L,2004,Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation IUBAB Life 56:449-456
Wilmut i,AE Schnieke J McWhir AJ Kind and KH Campbell,1997.viable offspring derived from fetal and adult mammalian cells Nature,385:810-813.

 
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