59 PRODUCTION OF HANDMADE CLONED GOAT EMBRYOS WITH TWO TYPES OF DONOR CELLS AND CULTURED IN THREE MEDIA

2010 ◽  
Vol 22 (1) ◽  
pp. 187
Author(s):  
M. K. Jena ◽  
D. Malakar ◽  
A. K. De ◽  
S. Garg ◽  
Y. S. Akshey
Keyword(s):  
Electric Pulse ◽  
Donor Cell ◽  
Single Pulse ◽  
Pcr Analysis ◽  
Fibroblast Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Arcsine Transformation ◽  
Agar Gel Electrophoresis ◽  
Adult Fibroblasts

The study was carried out to see the developmental efficiency of handmade cloned goat embryos with 3 different media: RVCL (Research Vitro Cleave, Cook, Brisbane, Australia), EDM (Embryo Development Media) and modified SOF (mSOF) and 2 types of donor cells: fetal fibroblast and adult fibroblast. Oocytes were isolated from abattoir goat ovaries, matured in maturation medium, and incubated in 5% CO2 in air at 38.5°C for 24 h. Then, the oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona free by pronase (2 mg mL-1). Protrusion cone formation in oocytes was found 95 to 100% in T20 (TCM-199 + 20% FBS). The zona-free oocytes were bisected with an ultra-sharp micro blade on the basis of visible protrusion cones on the surface of oocytes using T20 medium containing 2.5 μg mL-1 cytochalasin-B. Fetal and adult fibroblast cells were used from confluent monolayer at passage 5 after trypsinizing in 0.25% trypsin-EDTA. One somatic cell was attached with one enucleated demioocyte by phytohemagglutinin and further fused with another enucleated demioocyte through electric pulse with a combination of alternating current (4 V) and direct current (2.10 kV cm-1 for 5 μs with a single pulse) in fusion medium (0.3 M mannitol, 0.1 mM MgCl2, 0.05 mM CaCl2, and 3 mg mL-1 BSA). Then, triplets were chemically activated with 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in the 3 media. Cleavage and morulae formation were observed at Day 7 from 183 triplets with fetal fibroblasts as donor cells in media RVCL (78.60 ± 2.23, 38.97 ± 2.1), mSOF (72.62 ± 1.89, 33.81 ± 1.9), and EDM (73.96 ± 1.66, 26.20 ± 2.04), respectively. Simultaneously, cleavage and morulae formation were observed at Day 7 from 203 triplets with adult fibroblasts as donor cells in media RVCL (73.97 ± 3.57, 33.14 ± 2.68), mSOF (76.22 ± 4.36, 26.15 ± 0.99), and EDM (65.97 ± 3.11, 20.78 ± 2.77), respectively. Among the 3 media, morulae formation was significantly higher in RVCL. Hence, in the subsequent experiment, RVCL medium was used exclusively in culture for 172 triplets. Cleavage and morulae formation at Day 7 was not significantly different (P < 0.05) in 2 types of donor cells; fetal fibroblasts (77.46 ± 3.65, 38.70 ± 2.66) and adult fibroblasts (75.74 ± 3.04, 33.77 ± 1.43), respectively. The data were analyzed using SYSTAT 7.0 (Systat, SPSS Inc., Chicago, IL, USA) after arcsine transformation, one-way ANOVA followed by Fisher’s LSD test. PCR analysis was performed with highly polymorphic 286-bp fragment of MHC-II DRB gene of cloned embryo and its donor cell. Similar bands were observed in both the cloned embryos and fibroblast cells in agar gel electrophoresis. In conclusion, development of handmade cloned embryos was higher in RVCL medium compared with the other two media tested, and efficiency of morulae formation was similar in both types of donor cells. Further study is required to optimize blastocyst production.

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Ying Liu ◽  
Olga Østrup ◽  
Juan Li ◽  
Gábor Vajta ◽  
Lin Lin ◽  
...  
Keyword(s):  
Cell Membranes ◽  
Time Window ◽  
Donor Cell ◽  
Egg Extract ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Pre Treatment ◽  
Adult Fibroblasts

SummaryPre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.

294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT

2011 ◽  
Vol 23 (1) ◽  
pp. 244
Author(s):  
R. Dutta ◽  
D. Malakar ◽  
K. Khate ◽  
J. Akshay
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Patient Specific ◽  
Fibroblast Cells ◽  
Donor Cells ◽  
Adult Fibroblasts

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance. The authors acknowledge N.D.R.I for the financial and infrastructural assistance.

scholarly journals Cats cloned from fetal and adult somatic cells by nuclear transfer

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 245-249 ◽  
Author(s):  
X J Yin ◽  
H S Lee ◽  
Y H Lee ◽  
Y I Seo ◽  
S J Jeon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Somatic Cells ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Skin Cells ◽  
Fetal Fibroblasts ◽  
Adult Skin

This work was undertaken in order to study the developmental competence of nuclear transfer (NT ) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.

scholarly journals 4CHROMOSOMAL STABILITY OF AFRICAN WILD CAT (FELIS SILVESTRIS LIBICA) SOMATIC CELLS AND CLONED EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 124 ◽  
Author(s):  
A.M. Giraldo ◽  
M.C. Gomez ◽  
B.L. Dresser ◽  
R.F. Harris ◽  
A.L. King ◽  
...  
Keyword(s):  
Chromosome Numbers ◽  
Chromosomal Abnormalities ◽  
Somatic Cells ◽  
Donor Cell ◽  
Domestic Cat ◽  
Fibroblast Cells ◽  
Metaphase Chromosomes ◽  
Skin Sample ◽  
Reconstructed Embryos ◽  
Donor Cells

Nuclear transfer (NT) procedures are generally inefficient and chromosomal abnormalities have been suggested as one causative factor. Embryos derived by somatic cell NT show a high incidence of aneuploidy, which seems to be reflective of the donor cell line with high chromosomal abnormalities (Bureau et al., 2003, Theriogenology 59, 239). Also, aneuploidies in some cell lines increase progressively with the number of passages (Denning et al., 2001, Cloning 3, 221–231). Therefore, the aim of the present study was to analyze the chromosomal stability of donor cells at different passages as well as that of reconstructed embryos derived from these cells. A primary culture of African wild cat (AWC) fibroblast cells was established from a skin sample and cultured until cells stopped dividing at passage 9. Chromosome numbers were determined in cells using a karyotyping technique (Iwasaki et al., 1992 J Exp Zool 261, 79–85) at passages 1 and 3 through 9. At passages 1, 3, 4 and 9, NT of AWC fibroblast cells into domestic cat cytoplasts was done (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Reconstructed blastocysts were treated with colcemid (0.28μgmL−1) and fixed for karyotyping to evaluate chromosome numbers in the blastomeres. Blastomere metaphases were often highly contracted or overlapping, so that only 1 to 7 sets of metaphase chromosomes per embryo were spread sufficiently to allow determination of ploidy. No attempt to produce NT embryos using cells at passages 5 through 8 was done because the percentages of aneuploidies of these passages were not significantly different (P&gt;0.05). Data were analyzed by chi-square test (P&lt;0.05). As shown in the Table, the percentage of aneuploidy in the somatic cells increased progressively with duration of culture. Furthermore, the percentage of chromosomal abnormalities in reconstructed embryos was similar to that of the cells from which they were derived. Accordingly, for future NT, we propose to use donor cells at early passages when the percentage of cells with chromosomal abnormalities is still relatively low. Research was funded partially by the John &amp; Shirley Davies Foundation. Chromosomal abnormalities in AWC cells and cloned embryos

scholarly journals 33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 139
Author(s):  
S.R. Cho ◽  
W.J. Son ◽  
C.S. Park ◽  
S.Y. Choe ◽  
G.J. Rho
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Group 1

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P&lt;0.05). Blastocyst development of cloned embryos was significantly (P&lt;0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

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.

46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 180
Author(s):  
F. F. Bressan ◽  
M. S. Miranda ◽  
F. Perecin ◽  
T. H. C. De Bem ◽  
M. Bajgelman ◽  
...  
Keyword(s):  
Genetically Modified ◽  
Cell Lineage ◽  
Donor Cell ◽  
Pregnancy Rates ◽  
Control Group ◽  
Specific Cell ◽  
Nuclear Reprogramming ◽  
Developmental Potential ◽  
Donor Cells ◽  
Fetal Fibroblasts

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P > 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P < 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil.

409 EFFICIENCY OF CO-TRANSFECTION OF TRANSGENE AND Neor GENE INTO GOAT AND PIG FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 320
Author(s):  
Y. M. Shin ◽  
S. M. Chang ◽  
B. C. Kim ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Transfection Efficiency ◽  
Somatic Cells ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Fibroblast Cells ◽  
Drug Resistant ◽  
Fetal Fibroblast ◽  
Resistant Genes ◽  
G418 Selection ◽  
Fetal Fibroblasts

Transgenic animals can be generated by nuclear transfer with genetically modified somatic cells in which the essential procedure of transgene transfection is required. Most transgene vectors are constructed to contain transgene and drug-resistant genes to enrich for somatic cells in which transgene integration has occurred. However, construction of transgene vectors along with drug-resistant genes may not be easy, due to inappropriate restriction sites. Therefore, in this study, two separate constructs, human tPA cDNA fused to β-casein promoter sequence as a transgene vector and neomycin-resistant gene (Neor) driven by PGK promoter as a drug-selectable gene, were co-transfected into pig and goat fetal fibroblast cells to estimate the efficiency of transgene transfection following G418 selection. First, goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) were tested for G418 resistance with different concentrations of G418. The pertinent concentrations of G418 were 800 µg mL−1 for GFF and 200 µg mL−1 for PFF. The linearized tPA vector and Neor gene vector were co-transfected into goat fetal fibroblasts and pig fetal fibroblasts with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). The cells were selected following exposure of 800 µg mL−1 and 200 µg mL−1 G418 for GFF and PFF, respectively, for 14 days. Cell colonies surviving G418 selection were assayed by PCR amplification with tPA-specific primers. Initially 2 × 106 GFF and PFF were transfected. Resistant colonies were counted and transferred to 24-well plates for expansion and PCR analysis. The results of co-transfection experiments are summarized in Table 1. The transfection of 2 × 106 GFF and PFF yielded an estimated 96 and 93 colonies, respectively, which survived as the G418 selection. However, 54 colonies of GFF and 39 colonies of PFF proliferated during expansion and were subjected to PCR analysis. Twenty-three and 5 of these colonies were identified to contain tPA transgene in GFF and PFF colonies, respectively. Transfection frequencies for tPA gene were 42.6% and 12.8% in GFF and PFF, respectively. These results suggest that co-transfection of transgene vector with Neor gene can be an alternative method for transfection of transgenes into fetal fibroblast cells. Table 1. Transfection efficiency of goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) following co-transfection of tPA gene and Neor gene

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