Showing papers on "Cloning published in 2005"
TL;DR: The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. putida and Yersinia pestis, and 'GFP-tagging' Y. pestis.
Abstract: For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.
702 citations
TL;DR: The recent advent of recombinational cloning techniques has significantly improved the ability to investigate gene functions systematically, and novel binary T-DNA vectors are now available to assemble multiple DNA fragments simultaneously, which greatly facilitate plant cell and protein engineering.
574 citations
TL;DR: The ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.
Abstract: Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.
424 citations
TL;DR: It is shown that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT and postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.
156 citations
01 Jan 2005
TL;DR: A chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3 was developed and three previously uncharacterized genome-specific satellite repeats were discovered in the centromeres of O. brachyantha and Oryza rhizomatis.
Abstract: The functional centromeres of rice (Oryza sativa, AA genome) chromosomes contain two key DNA components: the CRR centromeric retrotransposons and a 155-bp satellite repeat, CentO. However, several wild Oryza species lack the CentO repeat. We developed a chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3. Chromatin immunoprecipitation cloning was carried out in the CentO-less species Oryza rhizomatis (CC genome) and Oryza brachyantha (FF genome). Three previously uncharacterized genome-specific satellite repeats, CentO-C1, CentO-C2, and CentO-F, were discovered in the centromeres of these two species. An 80-bp DNA region was found to be conserved in CentO-C1, CentO, and centromeric satellite repeats from maize and pearl millet, species which diverged from rice many millions of years ago. In contrast, the CentO-F repeat shows no sequence similarity to other centromeric repeats but has almost completely replaced other centromeric sequences in O. brachyantha, including the CRR-related sequences that normally constitute a significant fraction of the centromeric DNA in grass species.
156 citations
TL;DR: Data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals.
Abstract: Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.
124 citations
TL;DR: The objectives of this paper are to review some of the successes and failures of the nuclear transfer procedure since the production of Dolly, and discuss studies from the laboratory and others which have modified the procedure in ways which may impact on development.
Abstract: It is now 8 years since the birth of Dolly, the first animal produced by nuclear transfer using a donor cell population established from an adult animal. During this time, the technique of nuclear transfer has been successfully applied to a range of mammalian species for the production of offspring using a plethora of donor cell types derived from both foetal and adult tissues. In addition, when coupled with genetic manipulation of the donor cells, transgenic offspring have been produced with a range of genetic modifications including gene knockouts and gene knockings. Despite the apparent successes of the technology, the efficiency of development to live offspring has remained low and developmental abnormalities still occur. The objectives of this paper are to review some of the successes and failures of the nuclear transfer procedure since the production of Dolly. In particular, we will review the major steps in the procedure and discuss studies from our laboratory and others which have modified the procedure in ways which may impact on development.
108 citations
TL;DR: The isolation and characterization of the isocyanide-containing eDNA-derived antibiotic 1, its biosynthetic genes (isnA and isnB), the origin of the amine nitrogen atom, and the general outline of the biosynthesis of 1 are reported, which results in a triplet resonance for the carbon atoms, which is a hallmark of this functional group.
Abstract: Cultured soil bacteria have been an important source of biologically active, naturally occurring small molecules. Uncultured soil bacteria, which outnumber their cultured counterparts by at least two orders of magnitude, are likely to be an equally important source of such molecules. In an attempt to access the biosynthetic potential of uncultured soil bacteria, we have explored an approach that involves the cloning and heterologous expression of DNA extracted directly from environmental samples (environmental DNA, eDNA) into readily cultured bacteria. Earlier, we described the identification and characterization of small-molecule antibiotics from antibacterially active eDNA clones found using a high-throughput phenotypic screen. This approach directly couples the biosynthesis of each natural product that is found to a relatively small piece of cloned eDNA and therefore permits the characterization of both new natural products and their biosynthetic gene clusters simultaneously. Herein, we report the isolation and characterization of the isocyanide-containing eDNA-derived antibiotic 1, its biosynthetic genes (isnA and isnB), the origin of the isocyanide nitrogen atom, and the general outline of the biosynthesis of 1. CSLG18, the eDNA clone that produces 1, was found in a cosmid library constructed from blunt-ended gel-purified high-molecular-weight eDNA extracted directly from soil collected in Boston (MA, USA). The cosmid library was screened for antibacterially active clones by using a top agar overlay which contained Bacillus subtilis. Clones that produced a zone of growth inhibition in the overlayed B. subtilis lawn were recovered from the assay plates and tested for the production of extractable organic antibacterial activities. Bioassay-guided fractionation of the antibacterially active extract in ethyl acetate from cultures of CSLG18 led to the isolation of 1, an isocyanide-functionalized C3-substituted indole. The indole and trans olefin were readily inferred from standard 1D and 2D NMR spectroscopic experiments. The triplet resonance observed at Dd= 107.4 ppm in the C NMR spectrum suggested the presence of the isocyanide functionality as C–N coupling between the nitrogen atom and the adjacent carbon atom of the isocyanide group results in a triplet resonance for the carbon atoms, which is a hallmark of this functional group. The proposed structure of 1 was subsequently confirmed by single-crystal X-ray diffraction (Figure 1). Although the cis isomer of 1 has been obtained from cultured bacteria, to the best of our knowledge 1 has never been reported as a natural product.
108 citations
TL;DR: This is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT andIVF embryos in cattle.
Abstract: Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and α-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.
97 citations
TL;DR: Current scientific interest in embryonic stem cells is a logical step in the progress of these studies and holds the hope of providing important research tools as well as possible therapeutic applications.
Abstract: Perspectives in Biology and Medicine, volume 48, number 2 (spring 2005):211–28 © 2005 by The Johns Hopkins University Press WITH THE SEQUENCING of the human genome and our increasing knowledge of the molecular mechanisms of basic cell functions, we are entering an era of rapid advance in the field of developmental biology. Current scientific interest in embryonic stem cells is a logical step in the progress of these studies and holds the hope of providing important research tools as well as possible therapeutic applications. The ethical controversy surrounding cloning for biomedical research (CBR)1 and human embryonic stem cell (ES cell) research arises from the fact that to obtain these cells living human embryos must be disaggregated and destroyed. Many Americans oppose such embryo destruction, believing that there is an implicit dignity and inviolability in the individual continuity of a human life from fertilization to natural death. Many others, however, believe that the benefits of advances in biomedical science outweigh these moral concerns.
88 citations
TL;DR: It is demonstrated that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.
Abstract: In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.
TL;DR: Although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, it is recommended that nt ES cell lines be established.
Abstract: The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.
TL;DR: By addressing the unique requirements of cloned embryos, the entire process of reprogramming may be accelerated, thus increasing cloning efficiency.
TL;DR: Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.
Abstract: Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 mg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs. developmental biology, early development, embryo, gene regulation
TL;DR: This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.
Abstract: A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.
TL;DR: Recent data already available on bovine somatic clones of both sexes indicate that they have a zootechnical performance similar to non cloned animals and they are able to reproduce normally without the pathologies associated to cloning thus confirming that the deviations observed in clones are of epigenetic origin and not transmitted to the progeny.
Abstract: Recent evolutions of somatic cloning by nuclear transfer are reported, especially in the bovine species where potential applications are underway for biomedicine in association with transgenesis, or for agriculture by improving livestock. The overall efficiency of this biotechnology remains low in terms of viable offspring, but significant progress has been achieved on the different steps of the technique. However, the in vivo development of bovine blastocysts derived from somatic nuclear transfer is characterised by some important features that lead to the "cloning syndrome". Important losses occur during the peri-implantation period and further late foetal loss is observed in association with the Large Offspring Syndrome. About 60-70% of the cloned calves born survive normally to the adult stage and present an apparently normal physiology. Recent data already available on bovine somatic clones of both sexes indicate that they have a zootechnical performance similar to non cloned animals and they are able to reproduce normally without the pathologies associated to cloning thus confirming that the deviations observed in clones are of epigenetic origin and not transmitted to the progeny.
TL;DR: These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis.
Abstract: Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.
TL;DR: DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells and identified several chromosome regions in which gene expression was aberrantly controlled in the somatic clones.
Abstract: DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9-40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4-4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.
TL;DR: This is the first study to report that failure of implantation may be due to aberrant expression of genes in the preimplantation cloned embryo, which are crucial for the early regulation and differentiation of the placenta.
Abstract: Placental abnormalities and failed implantation are characterized phenotypes that occur in many species as a result of somatic cell cloning. This study examines a number of genes, critical for early placental development and reports aberrant expression patterns in a number of cloned bovine blastocysts, thus implicating a role of these genes in failed implantation. Messenger RNA (mRNA) expression of eight genes critical for early placental and preimplantation development including Acrogranin, Cdx2, Eomes, ErbB3, ERR2, Hand1, MRJ, and Rex1 were analyzed in single, in vivo, in vitro, and cloned bovine blastocysts (produced by hand-made cloning (HMC) and serial hand-made cloning (SHMC)) following complementary DNA (cDNA) amplification with a SMART cDNA synthesis kit. Aberrant expression of Acrogranin, Cdx2, and ERR2 was detected in a number of blastocysts produced by SHMC. Other genes, Eomes and Hand1, were not detectable in, in vivo bovine blastocysts, suggesting a differential expression pattern between bovine and murine embryos. A number of control marker genes including Oct4, IFN-tau, and PolyA were expressed in all single blastocysts analyzed. This is the first study to report that failure of implantation may be due to aberrant expression of genes in the preimplantation cloned embryo, which are crucial for the early regulation and differentiation of the placenta.
TL;DR: This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.
Abstract: Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.
TL;DR: The results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.
Abstract: The aim of this study was to identify a novel 1,4-β-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43 552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombina...
TL;DR: The role of mitochondria in cloned farm animals with emphasis on nucleo-cytoplasmic interactions and consequences for embryo development is reviewed, as the donor cell nucleus is typically transferred with surrounding cy toplasm including mitochondria which become destroyed in some but not all species.
TL;DR: The precise location of the S5n gene located on chromosome 6 is reported, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids.
Abstract: Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S5n gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S5n gene. In the first-pass mapping, the S5n gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S5 region. In the fine mapping, the S5 region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S5n gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S5 region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S5n gene.
TL;DR: This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.
Abstract: Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.
TL;DR: To study the link between FAS and cell respiration further, a yeast strain is created that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene.
Abstract: Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.
01 Jan 2005
TL;DR: This work has derived monoclonal mice from B and T cells and used the genetic rearrangements of the immunoglobulin and T cell receptor genes to retrospectively verify the differentiation state of the donor nucleus.
Abstract: The full-term development of sheep, cows, goats, pigs and mice has been achieved through the transfer of somatic cell nuclei into enucleated oocytes. Despite these successes, mammalian cloning remains an inefficient process, with a preponderance of reconstructed embryos failing at early- to mid-gestation stages of development. The small percentage of conceptuses that survive to term are characterized by a high mortality rate and frequently display grossly increased placental and birth weights. It is likely that inappropriate expression of key developmental genes may contribute to lethality of cloned embryos. One of the most interesting issues of nuclear cloning is the question of genomic reprogramming, i.e. whether successful cloning requires the resetting of epigenetic modifications which are characteristic of the adult donor nucleus. Processes such as X-inactivation and genomic imprinting are known to depend on epigenetic modifications of the genome. The classical nuclear transfer experiments with frogs have suggested that the source of the donor nucleus affects the phenotype of the clone. We have, using expression profiling, compared gene expression in clones derived from embryonic stem (ES) cells and from somatic donor cell nuclei and find substantial gene dysregulation. Our results suggest that faulty reprogramming is caused by the nuclear cloning procedure itself. In addition, the type of donor nucleus contributes to the abnormal expression pattern seen in cloned animals. One of the major unresolved issues has been whether nuclei of terminally differentiated cells can be reprogrammed by transfer into the oocyte. To address this question we have derived monoclonal mice from B and T cells and used the genetic rearrangements of the immunoglobulin and T cell receptor genes to retrospectively verify the differentiation state of the donor nucleus. Finally, we discuss our recent studies on the reprogramming of nuclei from terminally differentiated neurons and from cancer cells.
TL;DR: Despite occurrence of this transition in 100% of SCNT constructs, cloned embryo development remains poor and cloned embryos express numerous somatic cell characteristics, indicating that reprogramming is slow or incomplete.
TL;DR: This study produced transgenic rice plants by Agrobacterium-mediated transformation with a large- insert T-DNA containing a 92-kb region of the wheat genome and showed that rearrangements of the large-insert T- DNA, involving duplication, deletion and insertion, had occurred in all four lines.
Abstract: Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.
TL;DR: The introduction of donor cell nucleus into the oocyte derived from another individual leads to generation of hybridic (in terms of genetic mitochondrial material) reconstructed oocytes, which can have a certain influence on the genotype and phenotype identity of offspring produced as a result of cloning.
Abstract: Mitochondria are semiautonomous organelles that contain double-stranded (α-helix) circular DNA molecules (mtDNAs) of about 16300 to 16500 base pairs (bp). The mtDNA encodes only 13 proteins, 22 tRNAs and 2 rRNAs. Up to 95% of proteins involved in biogenesis and functions of mitochondria are encoded by the cell nucleus. The copy number of mitochondrial genome in a typical mammalian somatic cell ranges from approximately 2 × 103 to about 5 × 103, whereas the number of mtDNA molecules in a single mature (Metaphase II) oocyte is about 1.6 × 105 in mice, 2.5 × 105 in cattle and 3-8 × 105 in humans. In the procedure of somatic or embryo cell cloning (nuclear transfer), mitochondria of nuclear donor cells, together with the nucleus, are transplanted to the enucleated recipient oocyte (ooplast). Thus, the cloned embryo should harbour the mtDNAs from both the donor cell and the recipient oocyte. In cloned animals, mitochondria are inherited primarily from recipient oocytes, and mitochondria from donor cells appear to be rapidly eliminated during the first few cleavage divisions and are almost undetectable by the blastocyst stage. This selective segregation of the donor mitochondrial genome, which takes place in the preimplantation development of nuclear transfer-derived embryos, leads gradually to cellular mtDNA homoplasmy. Only in some cases does the mitochondria originating from both donor cells and recipient oocytes coexist (the so-called mtDNA heteroplasmy). When cloning both embryos and adult individuals, what is often forgotten is the presence in the cytoplasm of donorand recipient-cells of mtDNA. This contains small (approximately 0.01%) amounts of total cell genetic information, but it is different from information recorded in the nuclear DNA (99.99% of cellular genome). Thus, introduction of donor cell nucleus into the oocyte derived from another individual leads to generation of hybridic (in terms of genetic mitochondrial material) reconstructed oocytes, which can have a certain influence on the genotype and phenotype identity of offspring produced as a result of cloning. The “ideal” clone can be obtained only with donor cell nucleus transplanted into the oocyte originating 214 mtDNA IN SOMATIC AND EMBRYO CLONING OF MAMMALS 215 SAMIEC M. from the same individual. Therefore “ideal mammalian clones” can only be the clones of females, whose overall mitochondrial genome has a completely homogenouspattern of regulatory and coding nucleotide sequences of all cellular mtDNA copies in all the somatic and germ cell lines.
TL;DR: Differences in levels of transcripts of IGF-related genes in the bovine embryos produced by NT compared with IVF, Vivo, and PA are suggested.
Abstract: The low efficiency of animal production using somatic cell nuclear transfer procedures is considered to be the result of an incomplete reprogramming of donor cell nucleus, which leads to abnormal expression of developmentally important genes. The objective of this study was to determine the abundance of gene transcripts of insulin-like growth factor (IGF)-related genes in cloned bovine embryos reconstructed with somatic cells. Single embryos derived from nuclear transfer reconstructed with somatic cells (NT-SC) or embryo blastomeres (NTEM), in vitro fertilization (IVF), in vivo production (Vivo), and parthenogenetic treatment (PA) were analyzed. The relative abundance of mRNA was examined by real-time PCR. Transcripts of the IGF-1 receptor (r) and IGF binding protein (BP)-2 were detected in all embryos, regardless of origin. IGF-IIr and IGFBP-3 transcripts signals in NT-SC embryos were detected with significantly lower frequencies of 25 and 50%, respectively. Although IGF-Ir and IGFIIr transcript levels were not significantly different in NT-SC, NT-EM, IVF, Vivo, and PA embryos, the relative abundance in individual embryos indicated large variation in NT-SC. IGFBP-2 and IGFBP-3 levels were high in the Vivo embryos compared with NT-SC, NT-EM, IVF, or PA embryos. These results suggest differences in levels of transcripts of IGF-related genes in the bovine embryos produced by NT compared with IVF, Vivo, and PA.