Cloning sheep from cultured embryonic cells

1998 ◽  
Vol 10 (8) ◽  
pp. 615 ◽  
Author(s):  
D. N. Wells ◽  
P. M. Misica ◽  
A. M. Day ◽  
A. J. Peterson ◽  
H. R. Tervit
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Embryonic Cell ◽  
Farm Animals ◽  
Embryonic Cells ◽  
Embryo Survival ◽  
Fluid Medium ◽  
Embryonic Cell Lines

The production of transgenic farm animals will be greatly enhanced with the development of cultured cell lines that remain totipotent following nuclear transfer. Here, data are presented that demonstrate the generation of both male and female cloned lambs from two established embryonic cell lines. Cytoplasts derived from in vivo oocytes resulted in slightly greater development to blastocyst (24% v. 17%) and survival to term (7% v. 2%) compared with in vitro oocytes. There was no advantage in co-culturing cloned embryos with oviductal epithelial cells compared with synthetic oviductal fluid medium in terms of development to blastocyst (18% v. 31%) or survival to term (both 8%). Although the survival of cloned embryos immediately after transfer was high based on ‘biochemical’ pregnancy, 64–80% of embryos failed over the attachment phase with in vivo cytoplasts. Although the co-transfer of trophoblastic vesicles improved embryo survival to Day 35 (45% v. 25%), there was no difference at term. A high proportion of fetuses were lost during the last trimester (43%), resulting in 11% of embryos transferred developing to term using in vivo cytoplasts (12/112). Five lambs have survived and two rams are fertile. The current nuclear transfer process is inefficient and further research is needed to improve the development of healthy fetuses.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 165-171
Author(s):  
M. Kishi ◽  
R. Takakura ◽  
Y. Nagao ◽  
K. Saeki ◽  
Y. Takahashi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Cell Cycle Stage

SummaryIn the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.

Leishmania donovani and L. major: Cultivation in vitro in tick embryonic cell lines

1987 ◽  
Vol 63 (2) ◽  
pp. 240-242 ◽  
Author(s):  
M. Nyindo ◽  
A. Shatry ◽  
L.S. Awiti ◽  
M. Chimtawi ◽  
L.D. Hendricks
Keyword(s):  
Cell Lines ◽  
Leishmania Donovani ◽  
Embryonic Cell ◽  
Cultivation In Vitro ◽  
Embryonic Cell Lines

73 PROTEOMIC ANALYSIS OF THE MAJOR CELLULAR PROTEINS OF BOVINE TROPHECTODERM CELL LINES DERIVED FROM IVP, PARTHENOGENETIC, AND NUCLEAR TRANSFER EMBRYOS: REDUCED EXPRESSION OF ANNEXIN I IN NUCLEAR TRANSFER-DERIVED CELL LINES

2006 ◽  
Vol 18 (2) ◽  
pp. 145
Author(s):  
N. Talbot ◽  
A. Powell ◽  
T. Caperna ◽  
W. Garrett
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Triosephosphate Isomerase ◽  
Lipid Binding ◽  
Cellular Proteins ◽  
Phosphoglycerate Mutase ◽  
Calcium Dependent ◽  
Annexin I

Trophectoderm cell lines were established from 8-day in vitro cultured bovine embryos that were derived from the fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 2 V-, 16 NT-, 12 P-, and 13 IVF-derived cell lines were compared by 2-D gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. One-hundred and eighteen in common protein spots were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominently expressed cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, antioxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexin I and II. In comparative analysis of the 2-D gels, the NT-derived trophectoderm showed diminished annexin I expression in comparison to the IVF-derived trophectoderm. Since annexin I is abundantly expressed in the placenta and has functions important to the maintenance of placentation, the down-regulation of annexin I in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo

1997 ◽  
Vol 110 (1) ◽  
pp. 1-10 ◽  
Author(s):  
P. Dvorak ◽  
J.E. Flechon ◽  
E.M. Thompson ◽  
V. Horak ◽  
P. Adenot ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Receptor Binding ◽  
Embryonic Cell ◽  
Mesoderm Induction ◽  
Embryonic Cells ◽  
Rabbit Embryo ◽  
Migrating Cells

Several peptide growth factors, including members of the fibroblast growth factor (FGF) superfamily, are potential inducers of mesoderm in vertebrates. Receptor binding of basic FGF (FGF-2) is promoted by cell surface or extracellular matrix proteoglycans. The substantial biosynthesis of proteoglycans by embryonic cells (called embryoglycans) and their potential role as ligands for growth factor receptors led us to examine the role of embryoglycans that carry the developmentally regulated oligosaccharide epitope TEC 1, in the binding of FGF-2 to cultured rabbit inner cell masses (ICMs). Culture of isolated ICMs in the presence of FGF-2 gave rise to well delimited colonies with migrating cells at the periphery. In these cells, TEC 1 staining shifts from a punctate pattern over the entire membrane, to an apical, finely granular distribution with some internalization. This shift occurs after 96 hours in culture. Here we show that: (1) migrating cells are mesoderm-like in phenotype; (2) antibodies against TEC 1 blocked FGF-2 mediated differentiation in vitro; (3) antibodies against TEC 1 selectively blocked binding of FGF-2 to ectodermal receptors and, vice versa, the binding of TEC 1-specific antibodies to ectodermal cells can be competed by excess FGF-2; (4) the same switch in TEC 1 staining patterns was observed in vivo, between the day 7 and the day 9 rabbit embryo. These data suggest the involvement of defined species of embryonic cell surface epitopes in the regulation of FGF-2 receptor binding. Moreover, this proposed binding activity is temporally restricted to ectodermal cells and disappears early during differentiation. Thus, the apical TEC 1 redistribution can be considered as the earliest indicator of mesoderm formation.

scholarly journals Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniela Bebbere ◽  
Susanne E. Ulbrich ◽  
Katrin Giller ◽  
Valeri Zakhartchenko ◽  
Horst-Dieter Reichenbach ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Donor Cell ◽  
Farm Animals ◽  
Nuclear Reprogramming ◽  
Mtdna Depletion ◽  
Mitochondrial Dna Depletion

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Studies on the locomotion of primordial germ cells from Xenopus laevis in vitro

Development ◽  
1977 ◽  
Vol 42 (1) ◽  
pp. 149-161
Author(s):  
Janet Heasman ◽  
Tim Mohun ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Cell Movement ◽  
Embryonic Cell ◽  
Artificial Substrates ◽  
Embryonic Cells ◽  
Adult Kidney

The mechanism of embryonic cell movement is poorly understood. Primordial germ cells (PGCs) of the anuran amphibian Xenopus laevis migrate individually from their site of determination in the embryonic endoderm to their site of differentiation, in the developing gonad. PGCs have been isolated during their migratory phase from tadpoles, and their movement studied in vitro on a variety of natural and artificial substrates. On all artificial substrates used, including acid-washed glass, tissue-culture plastics, poly-L-Iysine-coated glass, and collagen, the PGCs move by amoeboid extrusion of hemispherical lobopodia. Several considerations make it unlikely that this is the mechanism employed in vivo. On living cellular substrates, e.g. monolayers of Xenopus laevis embryonic cells, adult kidney cells, and adult mesentery cells, PGCs become firmly attached and undergo phases of elongation and contraction. They move by elongation, coupled with the extrusion of filopodia, followed by waves of contraction, and ultimately by retraction of the trailing end of the cell. Evidence is presented that this is the mode of locomotion normally employed by PGCs in vivo.

Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique

2005 ◽  
Vol 17 (5) ◽  
pp. 573 ◽  
Author(s):  
R. Tayfur Tecirlioglu ◽  
Melissa A. Cooney ◽  
Ian M. Lewis ◽  
Natasha A. Korfiatis ◽  
Renee Hodgson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Fusion Efficiency

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.

scholarly journals 36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS

2005 ◽  
Vol 17 (2) ◽  
pp. 168 ◽  
Author(s):  
M.C. Gomez ◽  
C.E. Pope ◽  
L. Lyons ◽  
A. Cole ◽  
M. Lopez ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Implantation Rate ◽  
Domestic Cat ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Fetal Survival

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines. Table 1. Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients

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