32 CLONING OF ELITE QUARANTINE SNIFFING DOG BY SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 164
Author(s):  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
J. Choi ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Donor Cells ◽  
Wide Range ◽  
Sound Sensitivity

Somatic cell nuclear transfer (SCNT) in assisted reproductive technologies has been considered for the conservation of valuable or endangered animals. Dogs that were originally bred for hunting, such as beagles, have an exceptional ability to detect a particular smell from many others. For that reason, the beagles have been used to detect quarantine risk items from a wide range of goods in assorted luggage without scaring or disrupting the passengers. Though very useful and highly in need, elite quarantine sniffing beagles with excellent abilities are rare; much time, effort, and money are required in producing them. Here, we have applied SCNT for propagation of elite quarantine sniffing dogs to save time and economic burden. Ear fibroblasts from a 10-year-old adult male elite quarantine sniffing beagle were isolated and cultured in vitro as donor cells. For SCNT, in vivo-matured oocytes, obtained by flushing the uterine tubes of oocyte donors (mixed breed), were used. The oocytes were enucleated, microinjected with donor cells, fused by electrical stimulation, and activated chemically. Reconstructed oocytes were surgically transferred into the uterine tube of naturally synchronous recipient females. A total of 212 activated cloned embryos were transferred into 12 female recipient dogs and 4 recipients became pregnant. The 4 pregnant recipients delivered 4 pups through caesarean section or natural delivery, but 1 died right after birth and did not show an abnormality. Other live puppies exhibited normal phenotypes; their appearance was similar to that of the donor dog. All cloned pups were genetically identical to the donor dog and their mitochondrial DNA was from their oocyte donor dogs. When the cloned pups were 16 weeks old, we conducted a Volhard test, which is commonly used to describe the following puppy aptitudes: social attraction, following, restraint, social dominance, elevation dominance, retrieving, touch sensitivity, sound sensitivity, and sight sensitivity. Dog behavior data on differences in transcript abundance were analyzed by a general linear mixed model. The 3 cloned pups showed similar behavioral tendencies. The present study demonstrates that NT technique using donor cell derived from 1 elite quarantine sniffing dog is useful to produce a large number of quarantine sniffing dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, Nestlé Purina Korea, and TS Corporation.

scholarly journals Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 319-330
Author(s):  
Naoki Hirose ◽  
Sayaka Wakayama ◽  
Rei Inoue ◽  
Junya Ito ◽  
Masatoshi Ooga ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Activation Process ◽  
Oocyte Activation ◽  
Basic Study ◽  
Injection Method ◽  
Significant Difference

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

scholarly journals Plasma Steroid Dynamics in Late- and Near-term Naturally and Artificially Conceived Bovine Pregnancies as Elucidated by Multihormone High-resolution LC-MS/MS

Endocrinology ◽  
2014 ◽  
Vol 155 (12) ◽  
pp. 5011-5023 ◽  
Author(s):  
Helio A. Martins-Júnior ◽  
Fábio L. V. Pinaffi ◽  
Rosineide C. Simas ◽  
Adriana K. Tarouco ◽  
Christina R. Ferreira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Steroid Hormones ◽  
Sex Steroids ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Term Pregnancy ◽  
Near Term

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17β-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25)

2020 ◽  
Vol 160 (11-12) ◽  
pp. 688-697
Author(s):  
Sharmila Ghosh ◽  
Candice F. Carden ◽  
Rytis Juras ◽  
Mayra N. Mendoza ◽  
Matthew J. Jevit ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Congenital Abnormalities ◽  
Assisted Reproductive Technologies ◽  
De Novo ◽  
Reproductive Technologies ◽  
Balanced Translocation ◽  
Translocation Breakpoints ◽  
Normal Offspring ◽  
Embryonic Death

We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear transfer from the same donor were karyotyped because of fertility issues. A balanced translocation t(12q;25) was found in one, but not in the other clone. The findings underscore the importance of routine cytogenetic screening of breeding animals and animals produced by assisted reproductive technologies. These cases will contribute to molecular studies of translocation breakpoints and their genetic consequences in the horse.

scholarly journals Stem cell therapies and benefaction of somatic cell nuclear transfer cloning in COVID-19 era

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Birbal Singh ◽  
Gorakh Mal ◽  
Vinod Verma ◽  
Ruchi Tiwari ◽  
Muhammad Imran Khan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Drug Testing ◽  
Human Interaction ◽  
Patient Specific ◽  
Wide Range

Abstract Background The global health emergency of COVID-19 has necessitated the development of multiple therapeutic modalities including vaccinations, antivirals, anti-inflammatory, and cytoimmunotherapies, etc. COVID-19 patients suffer from damage to various organs and vascular structures, so they present multiple health crises. Mesenchymal stem cells (MSCs) are of interest to treat acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection. Main body Stem cell-based therapies have been verified for prospective benefits in copious preclinical and clinical studies. MSCs confer potential benefits to develop various cell types and organoids for studying virus-human interaction, drug testing, regenerative medicine, and immunomodulatory effects in COVID-19 patients. Apart from paving the ways to augment stem cell research and therapies, somatic cell nuclear transfer (SCNT) holds unique ability for a wide range of health applications such as patient-specific or isogenic cells for regenerative medicine and breeding transgenic animals for biomedical applications. Being a potent cell genome-reprogramming tool, the SCNT has increased prominence of recombinant therapeutics and cellular medicine in the current era of COVID-19. As SCNT is used to generate patient-specific stem cells, it avoids dependence on embryos to obtain stem cells. Conclusions The nuclear transfer cloning, being an ideal tool to generate cloned embryos, and the embryonic stem cells will boost drug testing and cellular medicine in COVID-19.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes

2013 ◽  
Vol 25 (8) ◽  
pp. 1142 ◽  
Author(s):  
Insung Hwang ◽  
Yeon Woo Jeong ◽  
Joung Joo Kim ◽  
Hyo Jeong Lee ◽  
Mina Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Canis Lupus ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fusion Rate ◽  
Domestic Dogs ◽  
Domestic Dog ◽  
Canis Lupus Familiaris ◽  
Cloning Efficiency ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature’s diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P < 0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones’ inheritance of maternal domestic dog mitochondrial DNA.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer

2015 ◽  
Vol 40 (1) ◽  
pp. 7-15
Author(s):  
Bo Fu ◽  
Liang Ren ◽  
Di Liu ◽  
Jian-zhang Ma ◽  
Tie-zhu An ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Live Donor ◽  
Donor Cells

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

322 POTENTIAL OF PRIMARY PORCINE KIDNEY CELLS FOR THE GENERATION OF TRANSGENIC PIGS VIA SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 308
Author(s):  
A. Wuensch ◽  
A. Richter ◽  
M. Kurome ◽  
B. Kessler ◽  
V. Zakhartchenko ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Homologous Recombination ◽  
Somatic Cell ◽  
Single Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Porcine Kidney ◽  
Cell Clones ◽  
Donor Cells ◽  
Additive Gene

The generation of genetically tailored pig models for biomedical research using somatic cell nuclear transfer (SCNT) is an efficient and precise approach, whereas the outcome is crucially dependent on the source of nuclear donor cells. Especially for site-directed mutagenesis by homologous recombination, including the generation of single cell clones, the demands on the target cells are high. Different primary cells used for SCNT have been tested for their efficiency in SCNT experiments, but further characterisation of the specific cell types, their morphology, proliferation, lifespan, and stability of karyotype is mostly lacking. We have evaluated the potential of 2 primary porcine kidney cell lines (PKC) isolated from juvenile pigs by a simple collagenase digestion and culture in collagen-coated dishes as cell source for SCNT, including their morphology, proliferation capacity, transfection efficiency, and capacity to support full-term development of SCNT embryos after additive gene transfer or homologous recombination. Single cell clones generated by subcloning of PKC at passage 3 showed different morphologies, proliferation rates, and lifespan, indicating that PKC culture is a mixed population of different types of fibroblasts and/or other cells types. The PKC could be maintained in culture for up to 71 passages without signs of senescence and decreased proliferation, exhibiting a stable karyotype containing 74% normal chromosome numbers (2N = 38) determined from metaphase spreads. In contrast, porcine fetal fibroblasts (PFF) and porcine ear fibroblasts (PEF) could be not be passaged more than 20 times. The calculation of growth curves at passage 4 to 5 showed that PKC exhibited a higher proliferation rate with a population doubling time of 16.6 to 18.4 h compared with PFF (23.2. h) and PEF (32.9 h). Furthermore the determination of the developmental competence after SCNT using PKC at passage 4 in 3 independent experiments and in vitro cultivation for 7 days resulted in a higher blastocyst rate (21%) compared with that in PFF (9.1%) and PEF (4.3%). The comparison of different transfection methods (lipofection, nanofection, conventional electroporation, nucleofection), using an expression vector for green fluorescent protein (GFP), showed that the NucleofectorTM technology gave the best results with transfection efficiencies of 70 to 89%, high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of stress. So far, around 150 cloned piglets using 18 different gene constructs have been produced using stable transfected PKC after additive gene transfer and targeting of 3 different loci. These findings demonstrate that among the 3 tested types of donor cells, PKC, PFF, PEF, primary PKC have outstanding potential for the production of genetically modified pigs by SCNT. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.

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