306 GENERATION OF HUMAN A20 GENE-TRANSGENIC PORCINE FETAL FIBROBLASTS FOR SOMATIC CELL NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 233 ◽  
Author(s):  
M. Oropeza ◽  
B. Petersen ◽  
N. Hornen ◽  
D. Herrmann ◽  
H. Niemann
Keyword(s):  
Endothelial Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Activation ◽  
Expression Vector ◽  
Cell Clone ◽  
Cell Clones ◽  
Fetal Fibroblasts ◽  
A20 Gene

The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 � 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo. We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20.

306 GENERATION AND CHARACTERIZATION OF HUMAN A20 TRANSGENIC PIGS BY SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 250 ◽  
Author(s):  
M. Oropeza ◽  
B. Petersen ◽  
E. Lemme ◽  
A. Lucas-Hahn ◽  
A. L. Queisser ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Activation ◽  
Transgenic Animals ◽  
Endothelial Cell Activation ◽  
Transgenic Pigs ◽  
Transgenic Cell ◽  
Cell Clones

Xenotransplantation is considered a solution to diminish the acute shortage of human organs. Although the hyperacute rejection occurring instantly after xenotransplantation can already be reliably controlled, the following immunological defense like the acute vascular rejection (AVR) remains a major hurdle for long-term survival of xenografts in porcine-to-primate organ transplantation. AVR is primarily characterized by endothelial cell activation with severe consequences on coagulation. The human A20 (hA20) gene exhibits antiapoptotic and anti-inflammatory properties in endothelial cells (Ferran C 2006 Transplantation 82, 36–40) and could thus prevent endothelial cell activation leading to AVR and xenograft destruction. Here, hA20-transgenic pigs were produced by somatic cell nuclear transfer (SCNT) in order to examine the ability of hA20-expressing tissues and organs to modulate the AVR. Two hA20-expression vectors, containing the promoters CAGGS or EF1-α in addition to a neomycin resistance cassette, were transfected into porcine fetal fibroblasts. Transfection was accomplished by electroporation, and the cell clones were selected with G418 (800 μg mL–1) for 14 days. Resistant cell clones were screened in PCR with hA20-specific primers. SCNT was performed as previously described (Hölker M et al. 2005 Cloning Stem Cells 7, 35–44). After 8 SCNT sessions with pCAGGSEhA20-transgenic cell clones, embryo transfer was carried out to 16 peripuberal recipients resulting in 12 pregnancies (75%). Sixteen fetuses were isolated after sacrificing the recipient sows, and 45 piglets were born. Six of 16 fetuses (37.5%) and 15 of 45 (33.3%) piglets were transgenic. Four SCNT sessions were completed with pEF1hA20-transgenic cell clones following embryo transfer to 6 sows and 5 pregnancies were established (83.3%). Five fetuses were isolated and 14 piglets born. Five of 5 fetuses (100%) and 9 of 14 (64.3%) piglets were transgenic. Expression analysis (RT-PCR and Northern blots) showed transcription of the hA20 gene in heart, muscle, and cultivated porcine aortic endothelial cells of the pCAGGSEhA20-transgenic animals. No transcription was detected in pEF1hA20-transgenic animals. Current results show that hA20-transgenic pigs develop physiologically compared to wildtype counterparts. Expressing tissues and organs were only found in pCAGGSEhA20-transgenic animals, which are now being tested regarding functionality of the hA20 transgene. We would like to thank Prof. Beyaert from the University of Ghent, Belgium, for providing us with the A20-expression vector pCAGGSEhA20.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

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.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

scholarly journals Metabolic programming of a Warburg effect-like phenotype in donor fibroblasts prior to somatic cell nuclear transfer

2017 ◽  
Author(s):  
◽  
Bethany Rae Mordhorst
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Fetal Fibroblasts ◽  
Pharmaceutical Agents ◽  
Vitro Development

Gene edited pigs serve as excellent models for biomedicine and agriculture. Currently, the most efficient way to make a reliably-edited transgenic animal is through somatic cell nuclear transfer (SCNT) also known as cloning. This process involves using cells from a donor (which may have been gene edited) that are typically grown in culture and using their nuclear content to reconstruct a new zygote. To do this, the cell may be placed in the perivitelline space of an enucleated oocyte and activated artificially by a calcium-containing media and electrical pulse waves. While it is remarkable that this process works, it is highly inefficient. In pigs the success of transferred embryos becoming live born piglets is only 1-3%. The creation of more cloned pigs enables further study for the benefit of both A) biomedicine in the development of prognosis and treatments and B) agriculture, whether it be for disease resistance, feed efficiency, gas emissions, etc. Two decades of research has not drastically improved the cloning efficiency of most mammals. One of the main impediments to successful cloning is thought to be due to inefficient nuclear reprogramming and remodeling of the donor cell nucleus. In the following chapters we detail our efforts to improve nuclear reprogramming of porcine fetal fibroblasts by altering the metabolism to be more blastomere-like in nature. We used two methods to alter metabolism 1) pharmaceutical agents and 2) hypoxia. After treating donor cells both methods were used in nuclear transfer. Pharmaceutical agents did not improve in vitro development of gestational survival of clones. Hypoxia did improve in vitro development and we are currently awaiting results of gestation.

93 A sheep model of sickle cell disease using CRISPR/Cas9 and somatic cell nuclear transfer

2021 ◽  
Vol 33 (2) ◽  
pp. 154
Author(s):  
I. V. Perisse ◽  
G. Almeida-Porada ◽  
C. D. Porada ◽  
K. L. White ◽  
I. A. Polejaeva
Keyword(s):  
Sickle Cell Disease ◽  
Somatic Cell ◽  
Sickle Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
The United States ◽  
Fetal Life ◽  
Cell Disease ◽  
Immune System Development ◽  
Fetal Fibroblasts

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, with more than 2 million people in the United States alone carrying the sickle gene. Approximately 100 000 of these people are homozygous and suffer from SCD. Worldwide, there are ∼4.4 million people with SCD. SCD is caused by a single A to T nucleotide replacement at the sixth codon of the β-globin gene, which results in the substitution of a valine for glutamate in the β-globin protein. This causes the resultant tetrameric haemoglobin molecule to be unstable and the red cells carrying this aberrant protein to “sickle,” decreasing the ability of these cells to carry oxygen. Sheep and humans exhibit a high degree of homology at the level of the genome. In addition, their anatomy, organ physiology, and immune system development closely parallel that of humans during fetal life. The ovine β-globin (HBB) gene shares 87.5% similarity with human HBB. Therefore, we hypothesised that the introduction of the “sickle” mutation in the sheep genome would lead to the SCD phenotype in sheep that could provide a valuable platform for evaluating prenatal and postnatal drug and gene therapies for this disease. In this study, we used a CRISPR/Cas9 gene-editing approach to introduce the SCD mutation into the sheep β-globin/HBB gene. We designed a single guide (sg)RNA targeting exon 1 of the sheep β-globin/HBB gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from IDT. Using the Lonza-4D-Nucleofector system, the Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs) along with 101-bp single-stranded oligodeoxynucleotides, flanking the sickle cell mutation to enable homology-directed repair. The transfected SFFs were then cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. After 2 days, DNA was extracted from one-third of the SFFs and the remainder were seeded individually into five 96-well plates by limited dilution. After 7 days of culture, individual colonies were expanded into 24-well plates and cultured for an additional 3 days. PCR-restriction fragment length polymorphism (RFLP) analysis using Image J software demonstrated a high rate of mutations (∼70%) by either indels or SCD mutation that led to the loss of the restriction enzyme site, which was further supported by the analysis of cell colonies. We isolated 59 single cell-derived SFF colonies and, based on PCR/RLFP assay, 31/59 (52%) of them contained biallelic mutations (either indels or point mutations) and were subsequently submitted for Sanger sequencing. The sequencing demonstrated that 3 colonies (9.6%) contained biallelic SCD mutations in the β-globin/HBB gene. These data demonstrate that we successfully introduced the SCD mutation into SFFs. These cells will be used in the production of the first large animal (sheep) SCD model by somatic cell nuclear transfer in fall of 2020. This research was supported by UAES project 1343 and by USDA/NIFA multistate research project W-4171.

108. INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IS DEPENDENT ON COMPATIBLE CYTOPLASMIC FACTORS AND MITOCHONDRIAL DNA

2010 ◽  
Vol 22 (9) ◽  
pp. 26
Author(s):  
Y. Jiang ◽  
R. Kelly ◽  
A. Peters ◽  
H. Fulka ◽  
D. A. Mitchell ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Copy Number ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Development ◽  
Nuclear Pore ◽  
Mtdna Copy Number ◽  
Cytoplasmic Factors ◽  
Fetal Fibroblasts

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities to analyze and understand nuclear-cytoplasmic interactions. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility, specifically mitochondrial DNA (mtDNA), on successful development. Transfer of somatic murine fetal fibroblasts into enucleated porcine oocytes resulted in extremely low blastocyst rates (0.4%); increased DNA strand breaks; deficient nuclear pore complex arrangements and increased aberrant karyokinesis than observed in porcine-porcine SCNT embryos. Using allele specific-PCR analysis, murine mtDNA was detected at ever-decreasing levels to the blastocyst stage, with peak levels being 0.14 ± 0.055% in 2-cell embryos. Furthermore, these embryos reduced total mtDNA copy number during preimplantation development in a manner similar to porcine embryos. Injecting mouse embryonic stem cell extract and mitochondria along with the murine donor cell into a mitochondria depleted porcine oocyte, increased blastocyst zona pellucida thinning and blastocyst rates significantly (0.4% vs 3.42%) compared to the non-supplemented iSCNT group. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout preimplantation development. At later stages of preimplantation development, they possessed 48.00% ± 17.38% murine mtDNA and exhibited a mtDNA copy number profile similar to murine embryos. Overall, these data demonstrate that the addition of species compatible cytoplasmic factors and mitochondrial DNA improve developmental competence of iSCNT embryos.

scholarly journals Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lucia Natalia Moro ◽  
Diego Luis Viale ◽  
Juan Ignacio Bastón ◽  
Victoria Arnold ◽  
Mariana Suvá ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Myostatin Gene ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Abstract The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

Sign in / Sign up

Export Citation Format

Share Document