82 EFFECT OF DONOR CELL TRANSFECTION EVENTS ON EMBRYO AND FETAL SURVIVAL IN CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 158
Author(s):  
D. F. Salamone ◽  
C. B. Santos ◽  
J. L. Barañao ◽  
L. Bussmann ◽  
J. Artuso ◽  
...  
Keyword(s):  
Cell Line ◽  
Large Scale ◽  
Uv Light ◽  
Donor Cell ◽  
Transgenic Animals ◽  
Female Fetus ◽  
In Vitro Development ◽  
Fetal Survival ◽  
Vitro Development

In a large-scale bovine cloning program intended to obtain transgenic animals, it is important to maximize the number of calves produced. The present experiment was designed to test the hypothesis that different transfection events of the same somatic cell line can affect embryo and/or fetal survival. A fetal cell line was established from a 75-day-old Jersey female fetus. It was used as control and was also transfected 3 times with the same protocol. They were named Transfection 1, 2, and 3. Genetically modified cells were produced and isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL-HEPES with 1 mg mL−1 bovine testis hyaluronidase. Metaphases were assessed, and oocytes were enucleated by visualization with Hoechst 33342 (5 µg mL−1) under UV light (<6 s). Donor cells from different treatments were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium with an atmosphere of 5% CO2 + 5% O2 + 90% N2. Development to blastocysts (Days 7 to 9) was recorded. Two blastocysts were transferred nonsurgically per recipient cow, and pregnancies at 30 days were determined by ultrasonography. All data were analyzed by chi-square test. In vitro development to blastocysts was similar in all treatment groups. One birth was obtained from the control. Four and 7 births were obtained from Transfections 1 and 3, respectively. Although Transfection 2 had good in vitro development, this treatment did not produce any pregnancy. This fact demonstrated that the transfection event provides an additional source of variability in obtaining live transgenic animals. Our results pointed out the necessity to monitor fetal survival by ultrasonography in order to detect as soon as possible any deficiencies in development introduced by transfection. Table 1.Effect of different transfection events of same line on embryo and fetal survival

scholarly journals 152 IN VITRO AND IN VIVO DEVELOPMENT OF BOVINE IVP EMBRYOS FOLLOWING SINGLE-CELL BIOPSY ON DAY 4

2005 ◽  
Vol 17 (2) ◽  
pp. 226
Author(s):  
K. Hartwich ◽  
B. Peachey ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh
Keyword(s):  
Single Cell ◽  
Fetal Loss ◽  
Commercial Application ◽  
Embryo Survival ◽  
Continue Development ◽  
In Vitro Development ◽  
Fetal Survival ◽  
Cell Embryo ◽  
Vitro Development

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO2 in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 106/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO2, 7% O2, 88% N2. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.

Roscovitine Treatment Improves Synchronization of Donor Cell Cycle in G0/G1 Stage and In Vitro Development of Handmade Cloned Buffalo (Bubalus bubalis) Embryos

2012 ◽  
Vol 14 (2) ◽  
pp. 146-154 ◽  
Author(s):  
Naresh L. Selokar ◽  
Monika Saini ◽  
Mushariffa Muzaffer ◽  
G. Krishnakanth ◽  
Ambika P. Saha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Donor Cell ◽  
Bubalus Bubalis ◽  
In Vitro Development ◽  
Vitro Development

Donor cell trichostatin A treatment improves the in vitro development of cloned goat embryos

2015 ◽  
Vol 124 ◽  
pp. 76-80 ◽  
Author(s):  
Yi Min Wang ◽  
Xiang Bin Ding ◽  
Xin Feng Liu ◽  
Yong Zhang
Keyword(s):  
Trichostatin A ◽  
Donor Cell ◽  
In Vitro Development ◽  
Vitro Development

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.

17 TREATMENT WITH MGCD 0103 IMPROVES THE IN VITRO DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2016 ◽  
Vol 28 (2) ◽  
pp. 138
Author(s):  
H.-Y. Zhu ◽  
L. Jin ◽  
Q. Guo ◽  
Y.-C. Zhang ◽  
X.-C. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Development ◽  
Single Donor ◽  
Or Groups ◽  
Humidified Air ◽  
Vitro Development

We use MGCD 0103 to test whether the treatment with this novel histone deacetylase inhibitor improves the in vitro development of porcine somatic cell NT (SCNT) embryos. Matured eggs were cultured in medium supplemented with 0.05 M sucrose and 0.4 μg mL–1 demecolcine for 1 h. Treated eggs with a protruding membrane were transferred to medium supplemented with 5 μg mL–1 cytochalasin B and 0.4 μg mL–1 demecolcine. Protrusions were then removed by aspirating with a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. Fused eggs cultured for 1 h were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days at 38.5°C in 5% CO2 humidified air. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, the SCNT embryos were cultured in medium supplemented with 0, 0.2, 2, or 20 μM MGCD 0103 for 24 h and then transferred to medium without MGCD 0103. In Experiment 2, SCNT embryos were cultured in medium supplemented with 0.2 μM MGCD 0103 for 0, 6, 24, or 48 h and then transferred to medium without MGCD 0103. As shown in Table 1, development to the blastocyst stage increased in SCNT embryos treated with 0.2 μM MGCD 0103 compared with the control or groups treated with 2 or 20 μM MGCD 0103 (25.51 v. 10.74, 3.53, 3.20%, respectively; P < 0.05). As shown in Table 1, treatment for 6 h with 0.2 μM MGCD 0103 significantly improved the rate of blastocyst formation compared with the control or groups treated for 24 or 48 h (21.17 v. 10.48, 19.23, 10.20%, respectively; P < 0.05). Our results suggested that 0.2 μM MGCD 0103 treatment for 6 h can improve in vitro developmental competence of porcine SCNT embryos. Table 1.In vitro development of pig SCNT embryos with different concentrations of MGCD 0103 for 24 h, and with 0.2 μM MGCD 0103 for different durations

scholarly journals 25COLD STORAGE OF TISSUES AS SOURCE FOR DONOR CELLS DOES NOT REDUCE THE IN VITRO DEVELOPMENT OF BOVINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 135 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
F. Ergin ◽  
H. Sagirkaya ◽  
H.O. Mercan ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Cell Line ◽  
Nuclear Transfer ◽  
Calf Serum ◽  
Calcium Ionophore ◽  
Penicillin G ◽  
In Vitro Development ◽  
Viable Cells ◽  
Vitro Development

So far, most calves have been cloned from live adult cows or fresh fetal samples. There are few reports on using cells from a dead mammal for nuclear transfer (NT). This study was conducted to investigate whether different kind of viable cells could be obtained from tissues stored in cold for different duration and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FCS), 50μgmL−1 sodium pyruvate, 1% v:v penicillin-streptomycin (10.000UmL−1 penicillin G, 10.000μgmL−1 streptomycin), 10ngmL−1 EGF, 0.5μgmL−1 FSH, and 5μgmL−1LH. First cell line (CC) was established from articular cartilage of the leg of a slaughtered cow stored at 0°C in a cold storage room for 48h. Second cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 24h. Tissues from articular cartilage and muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500μm for 25μs. After fusion, NT units were activated using a combination of calcium ionophore (5μM), cytochalasin D (2.5μgmL−1), and cycloheximide (10μgmL−1), and cultured for 7 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from articular cartilage and muscle of a cow carcass stored at cold temperature for 24 and 48h and these cells have ability to generate NT blastocysts at rates similar to that of the controls. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F Ergin is a volunteer young researcher. Table 1 In vitro development of NT embroys from different cell lines

scholarly journals The Effects of Donor Cell Type and Culture Medium on in vitro Development of Domestic Cat Embryos Reconstructed by Nuclear Transplantation

2001 ◽  
Vol 14 (8) ◽  
pp. 1057-1061 ◽  
Author(s):  
Mokhamad Fahrudin ◽  
Takeshige Otoi ◽  
Ni Wayan Kurniani Karja ◽  
Masako Murakami ◽  
Tatsuyuki Suzuki
Keyword(s):  
Culture Medium ◽  
Donor Cell ◽  
Domestic Cat ◽  
Nuclear Transplantation ◽  
Cell Type ◽  
In Vitro Development ◽  
Vitro Development ◽  
Donor Cell Type
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