37 EFFECT OF CELL MANIPULATION FOR PRODUCTION OF TRANSGENIC CELL LINES ON GOAT CLONING EFFICIENCY

2015 ◽  
Vol 27 (1) ◽  
pp. 111 ◽  
Author(s):  
L. T. Martins ◽  
S. Gaudencio Neto ◽  
L. H. Aguiar ◽  
C. E. M. Calderón ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Cell Lines ◽  
Donor Cell ◽  
Pregnancy Rates ◽  
Cell Manipulation ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Transgenic Cell ◽  
Biological Variables

The generation of transgenic cell lines through standard cell transfection/antibiotic selection procedures may have a negative effect on cell viability, which in turn may compromise SCNT cloning efficiency. The aim of this study was to evaluate goat cloning efficiency by using transfected and nontransfected and transgenic and nontransgenic somatic cells as nucleus donors. Skin fibroblast cells from 1 adult doe were subjected to transfection by electroporation with the pBC1-hGCase-Neo transgene cassette containing the human glucocerebrosidase gene sequence (hGCase), following antibiotic cell colony selection. Four distinct syngeneic donor cell types were used for cloning: (a) wild type (nontransfected, nontransgenic) control cells (C1) at low passage (P3), (b) transfected negative control (transfected, nontransgenic) cells (CT) at high passage (P8), and (c) 2 lines of transfected, transgenic cells (CA, CB) at high passages (P8 through P10). Donor cell cycles were synchronized by high confluence (<95%) and 24-h serum starvation. Cloning procedures were performed by standard micromanipulation procedures. Following membrane fusion after a 1.25 kV cm–1 DC pulse for 45 µs, reconstructed structures were incubated in cytochalasin B for 1 h, and then activated in ionomycin/6-DMAP. After 12 h of IVC in G-1TM medium (Vitrolife, Englewood, CO, USA), 1-cell stage cloned embryos were surgically transferred into the oviduct of synchronous recipient females. To ascertain herd fertility and health and adequate procedures for embryo manipulation, synchronization protocols, and surgical interventions, groups of control females were subjected to cervical AI or surgical transfer of in vivo-produced 1-cell stage goat embryos (ET). Pregnancy diagnosis was performed by ultrasonography on Day 23, with weekly examinations until term. Data were analysed by the χ2 test (P < 0.05), and are presented in Table 1. The transfection process and passage number did not appear to affect development, as no differences in pregnancy rates were observed between cloned groups, although results with control cells (C1 and CT) and with CA and CB lines were similar to and lower than the AI and ET groups, respectively. Loss rate after cloning was high (88.8%), which may be due to faulty reprogramming, as other procedural and biological variables involved in the cloning process were endorsed by pregnancy rates and term viable pregnancies observed in the AI and ET groups. Cloning using CA donor cells at P9 resulted in two liveborn kids, with one dying soon after birth. Both animals were confirmed by molecular analyses as hGCase transgenic clones. Table 1.Overall efficiency after AI, superovulation and embryo transfer (ET) or cloning by nuclear transfer (NT) using control (C1), sham-transfected (CT), and 2 transgenic (CA, CB) syngeneic fibroblast cells lines in goats This research was funded by FINEP.

221 SOMATIC CELL GOAT CLONING USING ALLOGENEIC OR SYNGENEIC TRANSGENIC CELL LINES

2014 ◽  
Vol 26 (1) ◽  
pp. 224
Author(s):  
L. T. Martins ◽  
L. H. Aguiar ◽  
C. E. M. Calderón ◽  
S. G. Neto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Donor Cell ◽  
Serum Starvation ◽  
Chi Square ◽  
Cell Stage ◽  
Primary Fibroblast ◽  
Skin Cell ◽  
Standard Procedures

The aim of this study was to compare the efficiency of goat cloning by using cell lineages from distinct transgenic backgrounds. Primary fibroblast skin cell cultures from 2 females (allogeneic), transgenic for the human lysozyme gene (hLZ), were established following standard procedures. Cells from one hLZ genotype were used for the establishment of 2 double transgenic syngeneic cell lines by cell transfection (Nucleofector®, Lonza, Germany) with transgene cassettes containing either the human glucocerebrosidase gene (hGC) and neomycin resistance gene, or the human lactoferrin gene (hLF) with no selection gene. The hGC-transfected hLZ cells were antibiotic-selected (G418, Sigma-Aldrich, St. Louis, MO, USA) until the isolation of positive cell colonies, whereas hLF-transfected hLZ cells were seeded onto 100-mm culture plates (100 cells/plate) to allow colony outgrowth from individual cells. Isolated colonies were screened by PCR using specific primers for each transgene (hGC or hLF) and for hLZ and GAPDH (controls). Positive cells from one hLZ-hGC and one hLZ-hLF colony were used for cloning at passage 9, whereas hLZ cells from the other genotype were at passage 4. Cells were synchronized by high confluence and 24 h of serum starvation. Goat cloning was performed according to standard procedures (Feltrin et al. 2012 Reprod. Fertil. Dev. 25, 163). Briefly, cumulus-oocyte complexes from abattoir ovaries were in vitro-matured for 20 h. Oocyte enucleation and hLZ, hLZ-hGC, or hLZ-hLF donor cell insertion were done by micromanipulation. Reconstructed structures were fused by two 1.2-KV cm–1 DC pulses for 20 μs. Cloned embryos were cultured for 1 h in cytochalasin B and then activated in ionomycin/6-DMAP. After 12 h of in vitro culture in G-1™ medium (Vitrolife, USA), 1-cell stage embryos were transferred into the oviduct of synchronous females (Keefer et al. 2002 Biol. Reprod. 66, 199-203). Pregnancy diagnosis was performed by ultrasonography on Day 30, with weekly monitoring afterwards. Preliminary data from 6 replicates were analysed by the chi-square test (P < 0.05). Maturation rate and survival after enucleation were 42.8% (610/1425) and 72.9% (291/399), respectively. A total of 271 structures were reconstructed using the 3 donor cell lines. Fusion rates did not differ between hLZ (59.5%), hLZ-hGC (47.5%), and hLZ-hLF (48.5%) groups. A total of 68 hLZ, 92 hLZ-hGC, and 39 hLZ-hLF-derived embryos were transferred to 5, 7, and 3 recipients, respectively. No pregnancies were detected with the use of hLZ and hLZ-hLF cells. However, 3 pregnancies (one nonviable) were detected on Day 30 with hLZ-hGC cells (42.9%), with both viable pregnancies lost on Days 40 and 130 of gestation. Molecular analyses confirmed both concepti as transgenic clones from the hLZ-hGC cell line. In summary, antibiotic selection of positive colonies was effective at maintaining cell viability, with a positive response when used for cloning. Replications are in progress to evaluate the effect of cell colony isolation from individual cells (e.g. hLZ-hLF cells) on cell viability over time and on cloning outcome.

59 SYNCHRONIZATION OF CELL CYCLE STAGE OF BUFFALO (BUBALUS BUBALIS) FETAL FIBROBLAST CELLS BY DIFFERENT TREATMENTS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
N. L. Selokar ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
M. Muzaffar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bubalus Bubalis ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Cloning Efficiency ◽  
Fetal Fibroblast ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Major Factors

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal fibroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT. Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization Supported by grant No. 1(5)/2007-NAIP from ICAR, India.

26 HISTONE DEACETYLASE INHIBITOR SCRIPTAID IMPROVES EPIGENETIC REPROGRAMMING AND CLONING EFFICIENCY IN THE PIG

2015 ◽  
Vol 27 (1) ◽  
pp. 105
Author(s):  
S. Liang ◽  
T. Kim ◽  
N.-H. Kim ◽  
X.-S. Cui
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Histone H3 ◽  
Development Program ◽  
Donor Cell ◽  
Epigenetic Reprogramming ◽  
Republic Of Korea ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Deacetylase Inhibitor

After somatic cell nuclear transfer (SCNT), the epigenetic state of a differentiated donor cell nucleus must be reversed to the embryonic state. Incomplete epigenetic reprogramming and abnormal gene activation of the donor cell nuclei is thought to be the cause of low cloning efficiency. To improve cloning efficiency, we investigated the effect of scriptaid, a novel histone deacetylase inhibitor, on the in vitro development of porcine SCNT embryos were investigated. Cumulus cells collected from cumulus-oocyte complexes (COC) after 44 h of maturation were used for donor cell, and embryos were cultured in porcine zygote medium (PZM)-5 medium for 7 days. We found that treating SCNT embryos with 300 or 500 nM scriptaid for 20 h after activation increased developmental rate to the blastocyst stage (300 nM, 26.2%; 500 nM, 24.6% v. 100 nM, 18.3%; Ctrl, 15.7%; P < 0.05) and total cell numbers (300 nM, 43.5; 500 nM, 40.8 v. 100 nM, 33.8; Ctrl, 32.3; P < 0.05). Additionally, results of the TUNEL assay indicated that scriptaid decreased apoptosis (300 nM, 6.8% v. Ctrl, 11.4%; P < 0.05) in SCNT blastocysts. After the 300 nM scriptaid treatment, the levels of acetylated histone H3 lysine 9 and 5-hydroxymethylcytosines were increased (P < 0.05), and histone H3 lysine 9 trimethylation and 5-methylcytosine were decreased at the 1-cell stage, which might explain the enhanced (P < 0.05) transcript levels of mir-152, Oct4, Cdx2, and Bcl-xL and reduced (P < 0.05) transcription of Dnmt1, Casp3, and Bax in blastocysts. In conclusion, scriptaid enhances the developmental capacity by preventing apoptosis, and improves nuclear reprogramming in porcine SCNT embryos.This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ009601 and PJ009098), Rural Development Administration, Republic of Korea.

27 CLONING OF ADULT PIGS USING SCRIPTAID TREATMENT AND PREOVULATORY EMBRYO TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 120
Author(s):  
M. Albornoz ◽  
C. Colato ◽  
N. El-Beyrouthi ◽  
F. Mellano ◽  
A. Mellano ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Expected Time ◽  
Reconstructed Embryos ◽  
Adult Cell ◽  
Skin Biopsies

There is growing interest in the use of swine in biomedical research. Cloning from cultured somatic cells (SCNT) has been the preferred method to generate genetically modified swine models. In a recent report, swine cloning efficiency was increased by treatment of reconstructed embryos with the inhibitor of deacetylase enzymes Scriptaid (Zhao et al. 2010 Cel. Reprog. 12, 75). Also, the timing of SCNT-embryo transfer with respect to the recipient’s expected time of ovulation was shown to affect cloning efficiency, whereas preovulatory embryo transfer resulted in a higher rate of cloned piglets born compared to postovulatory embryo transfer (Petersen et al. 2008 Cloning Stem Cells 10, 355). Therefore, our objective was to combine Scriptaid treatment and preovulatory embryo transfer in the same protocol for swine cloning. Cumulus–oocyte complexes aspirated from 3- to 6-mm diameter follicles were matured in vitro under standard conditions (Martinez Diaz et al. 2010 Cel. Reprog. 12, 85) and used as host oocytes for SCNT. Fibroblast cell lines were established from skin biopsies collected from 2 adult boars and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Oocytes were micromanipulated in Tyrode’s lactate-pyruvate-HEPES medium supplemented with 7.5 μg mL–1 cytochalasin B (CB) and electrically fused using a single DC pulse of 1.6 kV cm–1 for 70 μs. Activation was performed using ionomycin (15 μM/5 min) followed by exposure to CB (7.5 μg mL–1) and cyclohexemide (10 μg mL–1) for 5 h in porcine zygote medium (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112). Reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Oocytes were then washed and cultured in PZM-3 medium until transfer. Peripubertal recipient gilts were synchronized by oral administration of altrenogest (Regu-Mate®; 20 mg day–1) for 12 days, followed by 1.000 IU eCG injected on the last day of altrenogest treatment and 500 IU hCG 72 h later. 1-cell stage embryos were transferred into the oviduct after ∼20 h from hCG injection or 22 h before the expected ovulation time. Pregnancy was confirmed and monitored by ultrasonography and parturition was induced by injecting PGF2α at Day 115 of pregnancy. A total of 840 reconstructed embryos were transferred into 10 gilts [average 84 (range 60–110) embryos/gilt]. 4 gilts (40%) were detected to be pregnant 4 weeks after transfer, and 2 (20%) delivered 1 (1100 g) and 2 (950 and 850 g) healthy cloned piglets. The number of embryos transferred to these 2 gilts was 85 and 70. These results confirm that Scriptaid treatment and preovulatory embryo transfer can be applied in the same cloning protocol to produce cloned piglets from adult cell lines. To our knowledge, these are the first cloned pigs produced in Latin America.

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 223-227 ◽  
Author(s):  
Gang Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Negative Effects ◽  
Cell Stage ◽  
Kunming Mouse ◽  
Donor Cells ◽  
Significant Difference ◽  
Developmental Rates

SummaryIn this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2–4, 5–7 or 8–10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p > 0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p > 0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p < 0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.

scholarly journals Effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer

Reproduction ◽  
2003 ◽  
pp. 535-542 ◽  
Author(s):  
X Li ◽  
JL Tremoleda ◽  
WR Allen
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Metaphase Ii Oocytes ◽  
Adult Fibroblasts

The effects of repeated passage in vitro of fetal fibroblast cells (FFC) and adult fibroblast cells (AFC) on nuclear remodelling and first embryonic division when used to reconstruct horse oocytes, and the reasons for the developmental block in progression to the two-cell stage were investigated. A total of 463 metaphase II oocytes produced 427 fibroblast-cytoplasm couplets after nuclear transfer, which finally resulted in 319 reconstructed oocytes. With increasing numbers of passages, the rates of nuclear remodelling decreased in both types of donor cell; about half of the fused donor cell nuclei showed the S-G2-prometaphase stages of the first embryonic division 18-20 h after cell-fusion treatment, irrespective of the number of donor cell passages (FFC: 49%; AFC: 53%). The rates of first embryonic division in the reconstructed oocytes fell with increasing age of the donor cells (FFC: 32%-26%-23%; AFC: 27%-23%-24%) and these rates were significantly lower than those obtained from metaphase II oocytes activated parthenogenetically (79%, P < 0.05). Microscopic analysis of the organization of the first embryonic division in the developmentally blocked oocytes reconstructed with either FFC or AFC showed that most of these (FFC: 78%; AFC: 92%) could not form the mitotic spindle and the metaphase plate of chromosomes. These findings indicate that either fetal or adult fibroblasts that have undergone relatively few passages in vitro are most suitable as donors. However, both types of cell have lower potential to restart first embryonic development after nuclear transfer than do the equivalent cells in other species. Improvement in the rate of donor cell nuclear progression from S-G2-prometaphase to beyond the metaphase stage, and the normal organization of first embryonic development in reconstructed horse oocytes, would seem to be the key to the production of cloned embryos in this species.

Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Hong-Bo Liu ◽  
Pei-Ru Lv ◽  
Xiao-Gan Yang ◽  
Xiao-E Qin ◽  
Dao-Yuan Pi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Mammalian Species ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Contact Inhibition ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
New Born ◽  
Reconstructed Embryos

SummaryMiniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10–14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10μg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

scholarly journals Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors

2014 ◽  
Vol 62 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Manita Wittayarat ◽  
Akira Fujiwara ◽  
Kaywalee Chatdarong ◽  
Mongkol Techakumphu ◽  
Yoko Sato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Fusion Rate ◽  
Cell Cycle Analysis ◽  
Control Group ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Chemical Inhibitors ◽  
M Phase

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 μg/mL roscovitine or 0.05 μg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

Targeted histone demethylation improves somatic cell reprogramming into cloned blastocysts but not postimplantation bovine concepti†

2020 ◽  
Vol 103 (1) ◽  
pp. 114-125
Author(s):  
Fanli Meng ◽  
Kathrin Stamms ◽  
Romina Bennewitz ◽  
Andria Green ◽  
Fleur Oback ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Mammalian Species ◽  
Donor Cell ◽  
Biologically Active ◽  
Cell Reprogramming ◽  
Serum Starvation ◽  
Cloning Efficiency ◽  
Major Barrier ◽  
Somatic Cell Reprogramming ◽  
Donor Cells

Abstract Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

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