SciCombinator

Discover the most talked about and latest scientific content & concepts.

Concept: Cloning

208

Somatic cell nuclear transfer (SCNT) is a unique technology that produces cloned animals from single cells. It is desirable from a practical viewpoint that donor cells can be collected noninvasively and used readily for nuclear transfer. The present study was undertaken to determine whether peripheral blood cells freshly collected from living mice could be used for SCNT. We collected a drop of peripheral blood (15-45 µl) from the tail of a donor. A nucleated cell (leukocyte) suspension was prepared by lysing the red blood cells. Following SCNT using randomly selected leukocyte nuclei, cloned offspring were born at a 2.8% birth rate. Fluorescence-activated cell sorting revealed that granulocytes/monocytes and lymphocytes could be roughly distinguished by their sizes, the former being significantly larger. We then cloned putative granulocytes/monocytes and lymphocytes separately, and obtained 2.1% and 1.7% birth rates, respectively (P > 0.05). Because the use of lymphocyte nuclei inevitably results in the birth of offspring with DNA rearrangements, we applied granulocyte/monocyte cloning to two genetically modified strains and two recombinant inbred strains. Normal-looking offspring were obtained from all four strains tested. The present study clearly indicated that genetic copies of mice could be produced using a drop of peripheral blood from living donors. This strategy will be applied to the rescue of infertile founder animals or a “last-of-line” animal possessing invaluable genetic resources.

Concepts: DNA, Gene, Genetics, Cell nucleus, Blood, Red blood cell, Cloning, Somatic cell nuclear transfer

172

Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors.

Concepts: Gene, Mutation, Viral vector, In vivo, In vitro, Cloning, Construction, Clone

166

Myostatin (MSTN) has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT) technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production.

Concepts: Genetics, Gene expression, Muscular dystrophy, Cloning, Livestock, Muscle hypertrophy, Somatic cell nuclear transfer, Myostatin

66

The health of cloned animals generated by somatic-cell nuclear transfer (SCNT) has been of concern since its inception; however, there are no detailed assessments of late-onset, non-communicable diseases. Here we report that SCNT has no obvious detrimental long-term health effects in a cohort of 13 cloned sheep. We perform musculoskeletal assessments, metabolic tests and blood pressure measurements in 13 aged (7-9 years old) cloned sheep, including four derived from the cell line that gave rise to Dolly. We also perform radiological examinations of all main joints, including the knees, the joint most affected by osteoarthritis in Dolly, and compare all health parameters to groups of 5-and 6-year-old sheep, and published reference ranges. Despite their advanced age, these clones are euglycaemic, insulin sensitive and normotensive. Importantly, we observe no clinical signs of degenerative joint disease apart from mild, or in one case moderate, osteoarthritis in some animals. Our study is the first to assess the long-term health outcomes of SCNT in large animals.

Concepts: Death, Osteoarthritis, Gerontology, Knee, Joint, Ageing, Cloning, Ligament

36

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Concepts: Genetics, Developmental biology, Stem cell, Stem cells, Cell biology, Cloning, Somatic cell nuclear transfer, Somatic cell

35

Previous studies of serial cloning in animals showed a decrease in efficiency over repeated iterations and a failure in all species after a few generations. This limitation led to the suggestion that repeated recloning might be inherently impossible because of the accumulation of lethal genetic or epigenetic abnormalities. However, we have now succeeded in carrying out repeated recloning in the mouse through a somatic cell nuclear transfer method that includes a histone deacetylase inhibitor. The cloning efficiency did not decrease over 25 generations, and, to date, we have obtained more than 500 viable offspring from a single original donor mouse. The reprogramming efficiency also did not increase over repeated rounds of nuclear transfer, and we did not see the accumulation of reprogramming errors or clone-specific abnormalities. Therefore, our results show that repeated iterative recloning is possible and suggest that, with adequately efficient techniques, it may be possible to reclone animals indefinitely.

Concepts: DNA, Gene, Genetics, Histone, Epigenetics, Histone deacetylase, Cloning, Somatic cell nuclear transfer

27

Cloned embryonic stem cells (ESCs) are the tools used for therapeutic cloning, which is designed to remedy disease, not as a means for reproduction. Thirteen years after the first successful derivation of cloned ESCs in mice, and eleven years after the first application of therapeutic cloning also in mice, these pluripotent cells have now been produced in humans. The work of Tachibana and colleagues (Cell, May 2013) shows that the cytoplasm of the human oocyte, like that of several other mammalian species, is endowed with reprogramming capacity after somatic cell nuclear transfer (SCNT). The developmental rates, up to 60% for blastocyst formation and up to 50% for ESC derivation, are very competitive in comparison with the alternative technology of the induced pluripotent stem cells (iPSCs). What did Tachibana and colleagues do differently from the other investigators whose previous attempts to produce cloned human blastocysts and ESCs were unsuccessful? The authors developed a highly refined method for oocyte enucleation and embryo culture, and meticolously selected the oocytes. What does the achievement of the cloned human ESCs mean for basic science and for biomedicine? The reprogramming factors of the human oocyte can be mined and transferred to other reprogramming systems, such as iPSC. Human iPSC’s reprogramming can now be tested against a natural reference-the human oocyte. The achievement of Tachibana and colleagues is a great leap forward for knowledge. It also raises questions, for instance the extent of potency of cloned human embryos, and some issues that society will likely have to confront, such as the principles that should guide the allocation of human oocytes for reproductive versus non-reproductive purposes.

Concepts: Embryo, Developmental biology, Stem cell, Stem cells, Embryonic stem cell, Induced pluripotent stem cell, Cloning, Somatic cell nuclear transfer

27

Recently in Cell, Mitalipov and colleagues report an advance that has eluded scientists for over a decade-the successful derivation of embryonic stem cell lines using somatic cell nuclear transfer, or SCNT (Tachibana et al., 2013).

Concepts: Developmental biology, Stem cell, Stem cells, Cell biology, Embryonic stem cell, Induced pluripotent stem cell, Cloning, Somatic cell nuclear transfer

27

AIMS: This study aims to establish a novel method for cloning GII norovirus genome using generic primers rationally designed based on multiple alignments of 96 GII norovirus genome sequences. METHODS AND RESULTS: Based on conservative analysis of 96 GII norovirus genome sequences available in GenBank, three fragments encompassing the full-length genome were rationally designed. Fragments A, B, and C were amplified by primers N1F/N2819R, N2689F/COG2R, and COG2F/Adaptor primer, respectively. Meanwhile, the sensitivity of the novel primers was evaluated, which could achieve 10(1) RTPCRU, as determined by the common detection primer pair JV12/JV13. The availability of the novel protocol was verified by sequencing two norovirus strains with different genotypes. CONCLUSIONS: Primers for GII norovirus genome clone were rationally designed, and a novel GII genome clone method was established. SIGNIFICANCE AND IMPACT OF THE STUDY: The three-fragment cloning method can be used as a universal tool to collect information on the genome of norovirus strains for future evolution and antivirus studies. This article is protected by copyright. All rights reserved.

Concepts: Gene, Genetics, Evolution, Sequence, Novel, Cloning, All rights reserved, Copyright

26

Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clones. This has returned the bottleneck in many of these technologies to the generation of DNA clones, and standard cloning techniques often dramatically limit the throughput and success of such processes. In order to overcome this bottleneck, higher-throughput and more parallel cloning processes need to be developed which would allow rapid, inexpensive production of final clones. In addition, there is a strong need to utilize standardized elements to avoid unnecessarily remaking fragments of clones that could be used in multiple constructs.The advent of recombinational cloning helped to increase the parallel processing of DNA clones, but was still limited by the need to generate different vector backbones for each specific need. The solution to this problem emerged with the introduction of combinatorial approaches to clone construction, based on either homologous or site-specific recombination processes. In particular, the Gateway Multisite system provides all of the necessary components for a highly parallel, inexpensive, rapid, and diverse platform for clone construction in many areas of proteomic and genomic research. Here we describe our optimized system for combinatorial cloning, including improvements in cloning protocols and construct design that permit users to easily generate libraries of clones which can be combined in parallel to create an unlimited number of final constructs. The system is capable of utilizing the tens of thousands of commercially available Gateway clones already in existence, and allows easy adaptation of most DNA vectors to the system.

Concepts: DNA, Gene, Genetics, Bioinformatics, Molecular biology, Cloning, Clone, Network performance