Showing papers in "Nature Biotechnology in 1998"

Multicolor molecular beacons for allele discrimination

Abstract: Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore. We found that their hairpin conformation enables the use of a wide variety of differently colored fluorophores. Using several molecular beacons, each designed to recognize a different target and each labeled with a different fluorophore, we demonstrate that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. A comparison of "hairpin probes" with corresponding "linear probes" confirms that the presence of the hairpin stem in molecular beacons significantly enhances their specificity.

Man-made cell-like compartments for molecular evolution

Abstract: Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10 7 -fold excess of genes encoding another enzyme.

Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation

Abstract: Whole-genome mRNA quantitation can be used to identify the genes that are most responsive to environmental or genotypic change. By searching for mutually similar DNA elements among the upstream non-coding DNA sequences of these genes, we can identify candidate regulatory motifs and corresponding candidate sets of coregulated genes. We have tested this strategy by applying it to three extensively studied regulatory systems in the yeast Saccharomyces cerevisiae: galactose response, heat shock, and mating type. Galactose-response data yielded the known binding site of Gal4, and six of nine genes known to be induced by galactose. Heat shock data yielded the cell-cycle activation motif, which is known to mediate cell-cycle dependent activation, and a set of genes coding for all four nucleosomal proteins. Mating type α and a data yielded all of the four relevant DNA motifs and most of the known a- and α-specific genes.

Molecular evolution by staggered extension process (StEP) in vitro recombination

Abstract: We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynu-cleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisin E.

DNA chips: state-of-the art

Abstract: The technology and applications of microarrays of immobilized DNA or oligonucleotides are reviewed. DNA arrays are fabricated by high-speed robotics on glass or nylon substrates, for which labeled probes are used to determine complementary binding allowing massively parallel gene expression and gene discovery studies. Oligonucleotide microarrays are fabricated either by in situ light-directed combinational synthesis or by conventional synthesis followed by immobilization on glass substrates. Sample DNA is amplified by the polymerase chain reaction (PCR), and a fluorescent label is inserted and hybridized to the microarray. This technology has been successfully applied to the simultaneous expression of many thousands of genes and to large-scale gene discovery, as well as polymorphism screening and mapping of genomic DNA clones.

Gene transfer into muscle by electroporation in vivo

Abstract: Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Applications of this method have been limited by the relatively low expression levels of the transferred gene. We investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice were injected with the plasmid DNA, and then a pair of electrode needles were inserted into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5 levels were assayed. Mice that did not receive electroporation had serum levels of 0.2 ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of muscles injected with a lacZ expression plasmid showed that in vivo electroporation increased both the number of muscle fibers taking up plasmid DNA and the copy number of plasmids introduced into the cells. These results demonstrate that gene transfer into muscle by electroporation in vivo is more efficient than simple intramuscular DNA injection.

Agrobacterium tumefaciens-mediated transformation of filamentous fungi.

Abstract: Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis. It is routinely used for the genetic modification of a wide range of plant species. We report that A. tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus. We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A. awamori protoplasts. The majority of the A. awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. The T-DNA integrated into the A. awamori genome in a manner similar to that described for plants. We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A. tumefaciens is generally applicable to filamentous fungi.

Engraftable human neural stem cells respond to development cues, replaceneurons, and express foreign genes

Abstract: Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

Role of morphogenetic proteins in skeletal tissue engineering and regeneration.

Abstract: Morphogenesis is the developmental cascade of pattern formation and body plan establishment, culminating in the adult form. It has formed the basis for the emerging discipline of tissue engineering, which uses principles of molecular developmental biology and morphogenesis gleaned through studies on inductive signals, responding stem cells, and the extracellular matrix to design and construct spare parts that restore function to the human body. Among the many organs in the body, bone has considerable powers for regeneration and is a prototype model for tissue engineering. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and has permitted the isolation of bone morphogens, such as bone morphogenetic proteins (BMPs), from demineralized adult bone matrix. BMPs initiate, promote, and maintain chondrogenesis and osteogenesis, but are also involved in the morphogenesis of organs other than bone. The symbiosis of the mechanisms underlying bone induction and differentiation is critical for tissue engineering and is governed by both biomechanics (physical forces) and context (microenvironment/extracellular matrix), which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion glycoproteins, including fibronectins and laminin. Rules of tissue architecture elucidated in bone morphogenesis may provide insights into tissue engineering and be universally applicable for all organs/tissues, including bones and joints.

Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection.

Abstract: Molecular chaperones protect proteins against environmental and physiologic stress and from the deleterious consequences of an imbalance in protein homeostasis. Many of these stresses, if prolonged, result in defective development and pathologies associated with a diverse array of diseases due to tissue injury and repair including stroke, myocardial reperfusion damage, ischemia, cancer, amyloidosis, and other neurodegenerative diseases. We discuss the molecular nature of the stress signals, the mechanisms that underlie activation of the heat shock response, the role of heat shock proteins as cytoprotective molecules, and strategies for pharmacologically active molecules as regulators of the heat shock response.

An efficient route to human bispecific IgG

Abstract: Production of bispecific IgG (BsIgG) by coexpressing two different antibodies is inefficient due to unwanted pairings of the component heavy and light chains. To overcome this problem, heavy chains were remodeled for heterodimerization using engineered disulfide bonds in combination with previously identified "knobs-into-holes" mutations. One of the variants, S354C:T366W/Y349'C:T366'S:L368'A:Y407++ +'V, gave near quantitative (approximately 95%) heterodimerization. Light chain mispairing was circumvented by using an identical light chain for each arm of the BsIgG. Antibodies with identical light chains that bind to different antigens were identified from an scFv phage library with a very restricted light chain repertoire for the majority (50/55) of antigen pairs tested. A BsIgG capable of simultaneously binding to the human receptors HER3 and cMpI was prepared by coexpressing the common light chain and corresponding remodeled heavy chains followed by protein A chromatography. The engineered heavy chains retain their ability to support antibody-dependent cell-mediated cytotoxicity as demonstrated with an anti-HER2 antibody.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Abstract: Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43–58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

Abstract: To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

Improving protein therapeutics with sustained-release formulations

Abstract: Although numerous protein therapeutics have been approved or are in advanced clinical testing, the development of more sophisticated delivery systems for this rapidly expanding class of therapeutic agents has not kept pace. The short in vivo half-lives, the physical and chemical instability, and the low oral bioavailability of proteins currently necessitate their administration by frequent injections of protein solutions. This problem can be overcome by use of injectable depot formulations in which the protein is encapsulated in, and released slowly from, microspheres made of biodegradable polymers. Although the first report of sustained release of a microencapsulated protein was more than 20 years ago, the instability of proteins in these dosage forms has prevented their clinical use. Advances in protein stabilization, however, have allowed development of sustained-release forms of several therapeutic proteins, and clinical testing of a monthly formulation human growth hormone is currently in progress. The obvious advantage of this method of delivery is that the protein is administered less frequently, sometimes at lower overall doses, than when formulated as a solution. More importantly, it can justify commercial development of proteins that, for a variety of reasons, could not be marketed as solution formulations.

Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis

Abstract: We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules--molecular beacons--and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.

Containment of herbicide resistance through genetic engineering of the chloroplast genome.

Abstract: Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000–10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

High level multiplex genotyping by MALDI-TOF mass spectrometry

Abstract: A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5´ bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.

Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA.

Abstract: Dendritic cells (DC) generated from the peripheral blood mononuclear cells of healthy individuals or from cancer patients transfected with carcinoembryonic antigen (CEA) mRNA stimulate a potent CD8+ cytotoxic T lymphocyte (CTL) response in vitro. DCs are effectively sensitized with RNA in the absence of reagents commonly used to facilitate mammalian cell transfection. RNA encoding a chimeric CEA/LAMP-1 lysosomal targeting signal enhances the induction of CEA-specific CD4+ T cells, providing a strategy to induce T-help that may be necessary to generate and/or maintain an optimal CD8+ CTL response in vivo. CEA RNA-transfected DCs also serve as effective targets in cytotoxicity assays, thus providing a general method for inducing, as well as measuring, CEA-specific CTL responses across a broad spectrum of HLA haplotypes.

Identification of expressed genes by laser-mediated manipulation of single cells

Abstract: We describe a rapid noncontact method for the capture of single cells or small tissue areas of any size or shape directly within the cap of a common microfuge tube. Prior to the laser-mediated transfer, the specimen is isolated by laser microbeam microdissection, forming a clear-cut gap around the selected area. Laser treatment does not impair subsequent RNA analysis. We have used this method to isolate a single cell from archival colon adenocarcinoma, and were able to detect point mutations within codon 12 of c-Ki-ras2 mRNA after nested RT-PCR analysis.

Improved properties of FLP recombinase evolved by cycling mutagenesis.

Abstract: The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolabilty of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.

Rate of reaction with nitric oxide determines the hypertensive effect of cell-free hemoglobin

Abstract: Administration of extracellular hemoglobin-based oxygen carriers often induces mild increases in blood pressure In order to test whether nitric oxide (NO) scavenging is responsible for the hypertensive effect, we constructed and tested a set of recombinant hemoglobins that vary in rates of reaction with NO The results suggest that the rapid reactions of oxy- and deoxyhemoglobin with nitric oxide are the fundamental cause of the hypertension The magnitude of the blood-pressure effect correlates directly with the in vitro rate of NO oxidation Hemoglobins with decreased NO-scavenging activity may be more suitable for certain therapeutic applications than those that cause depletion of nitric oxide

Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips

Abstract: Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

In vivo electrically mediated protein and gene transfer in murine melanoma.

Abstract: We show that efficient permeabilization of murine melanoma can be obtained in vivo by applying electric pulses. More than 80% of the cell population is affected as shown by the penetration of propidium iodide. A protein, (β-galactosidase, can be transferred and expressed into the cells by incorporating either the protein or a plasmid carrying the reporter gene with respective efficiencies of 20% and 4%. This is obtained by a direct injection of either the protein or the plasmid in the tumor, followed by the application of electric pulses with surface electrodes in contact with the skin. This approach is simple and safe to use, reproducible, and specific; moreover, it is potentially applicable to a wide variety of tissues, cell types, and animals.

Construction of YAC-based mammalian artificial chromosomes.

Abstract: To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

Development of transgenic yellow poplar for mercury phytoremediation.

Abstract: We examined the ability of yellow poplar ( Liriodendron tulipifera ) tissue cultures and plantlets to express modified mercuric reductase ( merA ) gene constructs. Mercury-resistant bacteria express merA to convert highly toxic, ionic mercury, Hg(ll), to much less toxic, elemental mercury, Hg(O). Expression of merA in transgenic plants might provide an ecologically compatible approach for the remediation of mercury pollution. Because the alteration of the bacterial merA gene sequence is necessary for high-level expression in Arabidopsis thaliana , yellow poplar proembryogenic masses (PEMs) were transformed with three modified merA constructs via microprojectile bombardment. Each construct was synthesized to have altered flanking regions with increasing amounts of modified coding sequence. All merA constructs conferred resistance to toxic, ionic mercury in independently transformed PEM colonies. Stability of merA transgene expression increased in parallel with the extent of gene coding sequence modification. Regenerated plantlets containing the most modified merA gene ( merA18 ) germinated and grew vigorously in media containing normally toxic levels of ionic mercury. The merA18 plantlets released elemental mercury at approximately 10 times the rate of untransformed plantlets. These results indicate that plants expressing modified merA constructs may provide a means for the phytoremediation of mercury pollution.

Efficacy of a food plant-based oral cholera toxin B subunit vaccine.

Abstract: Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GM1-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GM1-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

Intercellular delivery of functional p53 by the herpesvirus protein VP22.

Abstract: The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, ie, efficient delivery of genes and gene products We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy

The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids

Abstract: Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.

Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells

Abstract: We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.