scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

24 EFFECT OF DONOR CELL TYPE ON IN VITRO AND IN VIVO DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
N. L. Selokar ◽  
P. Sharma ◽  
D. Kumar ◽  
R. K. Sharma ◽  
P. S. Yadav
Keyword(s):  
Somatic Cell ◽  
Seminal Plasma ◽  
Donor Cell ◽  
Cell Types ◽  
Developmental Competence ◽  
Cell Type ◽  
Donor Cells ◽  
Donor Cell Type

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
B. Mohana Kumar ◽  
H. F. Jin ◽  
J. G. Kim ◽  
S. A. Ock ◽  
H. J. Song ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Dna Hypomethylation ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Donor Cells ◽  
Fetal Fibroblasts

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P &lt; 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P &lt; 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P &lt; 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P &lt; 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 140
Author(s):  
N. Hornen ◽  
W. A. Kues ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
P. Hassel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Stem Cell Population ◽  
High Density Culture ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Normal Offspring

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (&lt;14 �m, 15–20 �m, &gt;21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.

scholarly journals Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽  
2001 ◽  
pp. 801-808 ◽  
Author(s):  
JL Liu ◽  
MK Wang ◽  
QY Sun ◽  
XR Zhang ◽  
LK Jiang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Donor Cell ◽  
Serum Starvation ◽  
Fresh Tissue ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

Effect of donor cell type, gender on efficiency of somatic cell nuclear transfer

2016 ◽  
Vol 231 ◽  
pp. S13
Author(s):  
Sezen Arat ◽  
Arzu Tas Caputcu ◽  
Mesut Cevik ◽  
Tolga Akkoc ◽  
Gaye Cetinkaya
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Type ◽  
Donor Cell Type

scholarly journals 36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 140
Author(s):  
Y. Echelard ◽  
E. Memili ◽  
S.L. Ayres ◽  
M. O'Coin ◽  
L.H. Chen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Reproductive Tract ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Student’S T

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts. Table 1

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