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Results for “Nuclear Transfer Techniques”

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PubMed2006

A double nuclear transfer technique for cloning pigs.

The first round of double nuclear transfer (NT) procedure includes the following steps: transfer of somatic cell nuclei into enucleated recipient oocytes, fusion, activation, and culture of reconstructed oocytes. The next day, a second round of NT is performed by removing karyoplasts from 1-d-old NT embryos and transferring them into in vivo-derived zygotes from which the two pronuclei have been removed. Couplets are then fused using an electrical pulse and transferred into synchronized recipient gilts. This system, which uses fertilized oocytes as cytoplast recipients, bypasses the inefficiencies of artificial activation procedures, and may promote more successful development.

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PubMed2003

Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations.

This study was conducted to improve a porcine somatic cell nuclear transfer (SCNT) technique by optimizing donor cell and recipient oocyte preparations. Adult and fetal fibroblasts, and cumulus and oviduct cells were used as donor cells, and in vivo- and in vitro-matured oocytes were employed as recipient oocytes. The percentages of fusion and development to the blastocyst stage, the ratio of blastocysts to 2-cell embryos, and cell number of blastocysts were monitored as experimental parameters. In Experiment 1, donor cells of four different types were transferred to enucleated oocytes matured in vitro, and more (P < 0.05) blastocysts were derived from SCNT of fetal fibroblasts than from that of other cells (15.9% versus 3.1-7.9%). For SCNT using fetal fibroblasts, increasing the number of subcultures up to 15 times did not improve developmental competence to the blastocyst stage (12.2-16.7%). In Experiment 2, fetal fibroblasts were transferred to enucleated oocytes that matured in vivo or in vitro. When parthenogenetic activation of both types of oocytes was conducted as a preliminary control treatment, a significant increase in blastocyst formation was found for in vivo-matured compared with in vitro-matured oocytes (36.4% versus 29.5%). However, no improvement was achieved in SCNT using in vivo-matured oocytes. In conclusion, the type of donor somatic cell is important for improving development after porcine SCNT, and fetal fibroblasts were the most effective among examined cells. A system with good reproducibility has been established using fetal fibroblasts as the donor karyoplast after subculturing 1-10 times, and using both in vivo and in vitro-matured oocytes as the recipient cytoplast.

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PubMed2005

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

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.

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PubMed2001

Comparison of gene transcription in cloned bovine embryos produced by different nuclear transfer techniques.

The efficiency of animal production using cloning technology is still relatively low and research to determine a more efficient nuclear transfer procedure is ongoing. One approach which may be informative in assessing the viability of nuclear transfer embryos is the analysis of embryonic gene expression. Using RT-PCR techniques we have previously detected the aberrant expression of FGF4, FGFr2 and IL6 in a significant proportion of bovine granulosa cell-derived nuclear transfer embryos, which correlated with a limited developmental potential in vivo. In order to analyse the effect of different donor cell nuclei on embryonic gene expression we have now analysed the expression of these genes in nuclear transfer embryos reconstructed with fetal epithelial cell nuclei. In addition, we have compared the expression of these genes in bovine nuclear transfer embryos produced by cell fusion or direct injection with variations in the timing of oocyte activation. In all nuclear transfer embryos analysed, FGFr2 and IL6 transcripts were detected at a similar rate to that in IVF embryos. However, the absence of FGF4 transcripts was again evident in a large proportion of nuclear transfer embryos and most significantly in those embryos whose development was activated almost immediately following the transfer of the donor nucleus. The results demonstrate the effects that different donor cell lines and different nuclear transfer procedures may have on the expression of developmentally important genes in nuclear transfer embryos.

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PubMed2006

Techniques for nuclear transfer to mouse embryonic stem cells.

In this chapter, methods are described that permit the enucleation of mouse embryonic stem (ES) cells and the transfer of donor nuclei to these cells before or after enucleation has taken place. The small size and high nucleus-to-cytoplasm volume ratio of ES cells poses a challenge to their enucleation. The first step describes the production of lines of larger, polyploid ES cells, which are more suited to enucleation than diploid ES cells. In a second step, a simple centrifugal enucleation technique is described that allows efficient bulk production of ES cell cytoplasts and karyoplasts. Finally, techniques for nuclear transfer to ES cells are described, involving either transfer of karyoplasts to cytoplasts or the formation of heterokaryons between donor and recipient cells followed by the selective elimination of the polyploid nucleus. These methods have potential applications in the generation of autologous, diploid pluripotent cells from donor somatic cells. Also, they provide a novel dynamic model for studying nucleocytoplasmic interactions in ES cells.

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PubMed2001

Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate).

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PubMed1999

Development of the techniques for nuclear transfer in pigs.

Nuclear transfer in pigs was developed in the late 1980's. The techniques were based on previous studies in frogs, mice and cattle. Within stage nuclear transfer, pronuclear exchange, was followed by the transfer of nuclei from cleavage stage embryos. While these have resulted in term development, many problems remain. Recently progress on the problem of inadequate oocyte activation has been made and now there can be a refocus on the other aspects of the nuclear transfer procedure. The emphasis in developing the cloning/transgenic technology is easily justified, not so much by the ability to produce genetically identical animals for production agriculture, but for the potential to use a cell line that can be genetically engineered prior to the nuclear transfer. Pigs with specific genetic modifications will have a great impact on production agriculture as well as human medicine.

Animals↗
PubMed2006

Production of transgenic-clone pigs by the combination of ICSI-mediated gene transfer with somatic cell nuclear transfer.

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

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PubMed2003

Generation of bovine transgenics using somatic cell nuclear transfer.

The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

Animals↗
PubMed2003

Creating genetically modified pigs by using nuclear transfer.

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture.

Animals↗
PubMed1998

Strategies for activating nuclear transfer oocytes.

The technique of nuclear transfer can have enormous applications in the fields of agriculture and biomedicine. This is especially true if a cell line that has been transformed can be used as a source of nuclei for the nuclear transfer. One major aspect of the nuclear transfer procedures is that of oocyte activation. Without oocyte activation the transferred nucleus would never progress to the first interphase. It is therefore of utmost importance that the oocyte be activated in a fashion that is as normal as fertilization. The inability to obtain development after artificial activation of pig oocytes has been a limiting factor in the application of the nuclear transfer technology. Recently, a number of techniques have been developed that result in blastocyst stage embryos after oocyte maturation in vitro and artificial activation. The theories behind normal oocyte activation are reviewed as well as a number of methods of artificial oocyte activation. It is anticipated that such a review will provide the basis for the development of additional methods that are as efficient, or more efficient, at activating the unfertilized oocyte.

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PubMed2001

[Somatic cloning in mammals].

Somatic cloning in mammals became possible due to refinement of the nuclear transfer technique consisting of using nuclear donor cells in GO or activating reconstituted oocytes few hours after nuclear transfer (post-activation). Sheep, goats, pigs, cattle and mice have been cloned this way; the latter two species - of both sexes. Cloning of transgenic mammals producing human therapeutic proteins in milk (or urine) can find application in pharmacology and medicine. Somatic cloning of a goat producing human antithrombine III in its milk has already been achieved.

Animals↗
PubMed2005

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells.

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.

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PubMed2001

Nuclear transfer in practice.

The technique of nuclear transfer (NT) allows the production of embryos, fetuses, and offspring from a range of embryonic, fetal, and adult derived cell types in a range of species. Successful development is dependent upon numerous factors, including type of recipient cell, source of recipient cell, method of reconstruction, activation, embryo culture, donor cell type, and donor and recipient cell cycle stages. The present review will discuss the uses of NT, the techniques presently available, and the factors affecting subsequent development.

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PubMed2002

Gene transfer into zebrafish by sperm nuclear transplantation.

A technique for fertilizing zebrafish eggs by injection of sperm nuclei is described. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults. If sperm nuclei are preincubated for 20 min with DNA encoding the green fluorescent protein, transgene expression can be detected in all cells of the embryo. The use of condensed sperm nuclei allows injection with a small bore pipette, which is critical for successful injection of the relatively small zebrafish egg. This technique enables the generation of ubiquitously expressing transgenic zebrafish directly by microinjection. Hence, experiments involving transgenic fish can be completed in days, without the need for growing and breeding founders. This technique may also be used to generate transgenic lines, as transgene expression was visible in the offspring of transgenic founders. The method described here is likely to be applicable to other teleosts, such as medaka and salmon.

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PubMed2002

Adult murine neurons: their chromatin and chromosome changes and failure to support embryonic development as revealed by nuclear transfer.

Fully differentiated neurons in adult mammalian brains do not divide; consequently, their metaphase chromosomes have never been examined. Here we report metaphase chromosome constitutions of cortical neurons in adult mice visualized by a nuclear transfer technique. We found that although some reconstructed oocytes cloned from neuronal nuclei have an apparently normal karyotype, the majority do not. Regardless of chromosome morphology, nuclei of adult neurons totally lack the ability to support embryonic development. These findings support the hypothesis that fully differentiated neurons in adult mammalian brains are genomically altered.

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