Nuclear Transfer Method Research Articles

P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P<0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P<0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P<0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A

CRISPR-mediated editing of β-lactoglobulin (BLG) gene in buffalo

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

P-197 Nuclear transfer with almost nonexistent mitochondrial carryover -a mouse model-

Abstract Study question Can the transfer of aggregated chromosomes (AC) as compared to spindle-chromosome transfer (SCT) reduce mitochondrial DNA (mtDNA) carryover and improve development rates? Summary answer The rate of mtDNA carryover after AC transfer was only 0.1%, which was much less than that of 2.0% after SCT. What is known already Nuclear transfer techniques such as SCT and pronuclear transfer have been applied to prevent mitochondrial diseases and to treat early embryonic arrest, however, the problem of mtDNA carryover has not yet been resolved. We previously reported on an AC transfer (ACT) method in humans which could overcome the above problem, however, consequent embryonic development rates have not been investigated in Japan due to legal restrictions and no animal study has been conducted, as the formation of ACs has not been found in any other species. Study design, size, duration Following our success in the creation of ACs in mouse oocytes using IBMX (3-Isobutyl 1-methylxanthine), a phosphodiesterase inhibitor, a mouse model of ACT was established. This was followed by a comparison of the rates of embryo development and mtDNA carryover in both ACT and SCT in over 100 oocytes. The distribution of mitochondria around SC and AC were also examined by confocal laser microscope (FV3000, Olympus, Japan). Participants/materials, setting, methods GV stage oocytes were collected from 8-12 week B6D2F1 mice. After GVBD, oocytes were incubated in KSOM medium, containing 3 mM IBMX, for 10-14 hours until AC is formed. ACs and SCs were injected into enucleated MII stage donor oocytes and then fertilized at the MII stage. Embryos were cultured for 5 days to compare the rates of embryo development. Enucleated ACs and SCs were subjected to real-time PCR, to analyze the carryover of mtDNA. Main results and the role of chance The rates of fertilization, embryo cleavage and blastocyst formation in AC oocytes without AC transfer (control), in ACT and in SCT were 60.7%, 55.9%, 13.2% (control), 82.5%, 80.8% and 30.8% (ACT), and 81.0%, 74.5% and 31.4% (SCT) respectively. The ACT group showed a significant increase in fertilization (P = 0.007), embryo cleavage (p = 0.008) and blastocyst formation (p = 0.034) rates, as compared to those in the control group. There were no significant differences between ACT and SCT regarding the rates of fertilization, embryo cleavage and blastocyst formation. The relative, real-time PCR showed that mtDNA carryover in the ACT group was 0.1% ± 0.07%, which was significantly lower than that in the SCT group of 2.0% ± 0.6% (p = 0.027). 3D confocal laser microscopy revealed that mitochondria were not localized around either SCs or ACs, as they were around pronuclei. Limitations, reasons for caution Although we succeeded in establishing a mouse model of ACT, validation in human oocytes is required to confirm the benefit of this method. Further experiments are required to confirm if healthy offspring can be obtained after ACT. Wider implications of the findings ACT could reduce the amount of mtDNA carryover and overcome heteroplasmy. As ACT improved the low embryo development rate caused by IBMX and had similar embryo development rates to SCT, the findings of this study may give rise to ACT being employed as a novel method for human nuclear transfer. Trial registration number not applicatable

MSTN modification in goat mediated by TALENs and performance analysis.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. It can inhibit the proliferation of myoblasts and serve as an important candidate gene for animal breed improvement. Mutations of the MSTN gene can cause extensive skeletal muscle hyperplasia and hypertrophy, resulting in "double muscle" symptoms. This leads to reduction of animal fat differentiation and increase of muscle content, thereby meeting the demand for quality consumption of animal meat in the market. In order to obtain a double-muscle phenotype using mutant MSTN gene in cloned goat, the goat MSTN gene was target-modified by TALENs. In this study, the TALENs expression vector was designed and constructed in the first exon sequence of the goat MSTN gene, which was then transfected into the goat fetal fibroblasts. The resistant cell lines were obtained by puromycin selection, and the cell lines with the MSTN gene mutations were analyzed by PCR and gene sequencing, thereby identifying the mutation type(s). The MSTN gene mutant cell lines were used as the nuclear donor cells in somatic cell nuclear transfer procedures in goats, and The morphological structure of the muscle tissue of the goats with MSTN gene mutations was analyzed by tissue section. The body weight of the cloned goats were monitored at different months of age, which provided the growth trend of their weight at different developmental stages. The results show that a total of 109 MSTN gene mutant cell lines were obtained. The mutation efficiency was 79.0% (109/138), of which 46 were biallelic mutations, accounting for 33.3% (46/138) of the total cell lines. Four MSTN gene mutant cell lines (1 biallelic homozygous mutation, 3 non-homozygous mutations) with good growth status were selected for somatic cell nuclear transfer in 12 recipients, of which 4 were pregnant by B-ultrasound at 30 days, indicating the a 33.3% (4/12) pregnancy rate. Two cloned goats were born at the end of the pregnancy. Sequencing analysis showed that there was no mutation in one allele of the M-1 cloned lamb, and the other allele harbored a 3 bp-deletion. The M-2 cloned lamb harbored a 1 bp base insertion in one allele of the MSTN gene, and a deletion of 13 bp in the other allele, resulting in mutations in both alleles and the loss of the protein-coding sequence of MSTN after the mutation site. In addition, the muscle fibers of cloned M-1 goats are tightly arranged and thick, and their monthly body weight is higher than that of normal wild-type goats. However, it is still consistent with the growth trend of normal wild-type goats and the M-1 goats can develop into healthy adults. In summary, this study showed that goat fetal fibroblasts with the multiple MSTN gene mutations were successfully obtained by TALENs technology, and cloned goats with mutant MSTN genes could be generated by somatic cell nuclear transfer method, thereby providing a technical foundation for the cultivation of the "double muscle" phenotype goats, and serving as a reference method for the preparation of other transgenic animals in the future.

Effect of xylazine, detomidine, medetomidine and dexmedetomidine during laparoscopic SCNT embryo transfer on pregnancy rate and some physiological variables in goats

BackgroundThe present study was conducted to determine if using α2-adrenergic agonists results in decreased stress levels (lower cortisol levels) in goats used for laparoscopic embryo [somatic cell nuclear transfer (SCNT)] transfer; and there is an effect on pregnancy rate when stress levels are lessened. Sixty healthy does aged 24 ± 4 months and weighing 30 ± 3 kg were used in experimental, prospective, randomized and blinded study. In this study, embryos were obtained by the Somatic Cell Nuclear Transfer (SCNT) method. Animals were randomly assigned to five groups: control (normal saline); xylazine (100 μg kg− 1); detomidine (50 μg kg− 1); medetomidine (20 μg kg− 1); and dexmedetomidine (5 μg kg− 1). Embryo transfer (through laparoscopic technique) began at 15 min and continued till 45 min post-treatment. Heart rate (HR), respiratory rate (RR), rectal temperature (RT), and ruminal motility were performed before (baseline) and after drug administration. Pregnancy detection was performed 38 days after embryo transfer.ResultsCompared to control, HR, RR and ruminal motility were significantly lower in α2-adrenergic agonists groups at 5–90, 15–60, and 5–120 min, respectively. Serum cortisol values significantly increased from baseline in the control group 45 min after drug administration (p = 0.001). At time points 45 and 60 min, serum cortisol concentration was significantly lower in α2-adrenergic agonists groups compared with the control. The pregnancy rate in control group (n = 4/12, 33.3%) was significantly lower than xylazine (n = 9/12, 75%; p = 0.041), detomidine (n = 10/12, 83.3%; p = 0.013), medetomidine (n = 9/12, 75%; p = 0.041) and dexmedetomidine (n = 10/12, 83.3%; p = 0.013); but no significant differences were observed among different α2-adrenergic agonists groups.ConclusionAlph2-adrenergic agonists were effective on increasing the pregnancy rate of recipient goats receiving cloned embryos. No significant differences were detected among different α2-adrenergic agonists.

Pregnancy and Calving Rates of Cloned Dromedary Camels Produced by Conventional and Handmade Cloning Techniques and In Vitro and In Vivo Matured Oocytes.

Despite practical implication of cloning in camelids, its broad application has been hampered by technical and biological problems. Method of somatic cell nuclear transfer (SCNT) and oocyte competence are the principal technical and biological factors, respectively, that determine the cloning efficiency. This study, therefore, investigated differential contributions of two SCNT methods [modified handmade cloning (mHMC) vs. conventional (cNT)] and two recipient oocyte sources [abattoir-derived (Vitro) vs. FSH-stimulated (Vivo)] on the efficiency of dromedary camel cloning. The mHMC method supported similar rates of fusion, cleavage, and total blastocyst development, compared to conventional NT (cNT) (94, 89.1, and 68.5% vs. 78.9, 92, and 73.5%, respectively) when Vivo oocytes are used. However, using Vitro oocytes, mHMC supported significantly higher rates for these criteria, except for the cleavage, compared to cNT (95.5, 76.2, 25.2% vs. 75.3, 76.7, and 13.9%, respectively). A total of seven clones were born from mHMC/Vitro (four calves), mHMC/Vivo (one calf), cNT/Vitro (one calf), and mHMC/Vivo&Vivo (one calf)-derived embryos with overall efficiencies of 31.9, 26.6, 20, and 30% for initial pregnancy, 10.6, 6.6, 7.5, and 5% for development to term, and 8.5, 6.6, 2.5, 5% for development to weaving, respectively. To conclude, the quality of recipient oocyte greatly impacts cloning efficiency in vitro with no apparent carrying over effect on cloning efficiency in vivo, but the efficiency of SCNT method may compensate for the initial poor oocyte competence during in vitro and in vivo development of cloned embryos. The introduced mHMC could be a superior alternative to conventional method for simple, fast, and efficient production of cloned offspring in camelids.

Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

Comparison of Developmental Efficiency of Murine Somatic Cell Nuclear Transfer Protocol

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher’s competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

Transfection of early and late stage sheep spermatogonial stem cells in culture

The genetic manipulation of spermatogonial stem cells (SSCs) can be used as an alternative to somatic cell nuclear transfer method for the production of transgenic animals. SSCs are now in vitro cultured and transplanted in sheep, however, there are no known protocol for DNA transfection of sheep SSCs. The aim of present study was to define the optimal transfection conditions of spermatogonial stem cells (SSCs) in early and late ovine SSC colony formation stages in culture. SSCs were isolated from the slaughterhouse ram testis tissue using a two-step enzymatic digestion process. Results showed that, 2 µl of DNA with 0.5 µg of Lipofectamin or Turbofect were able to transfect SSC colonies at late stage. Since the colonies of SSCs were not in logarithmic growth phase, around 15% of colonies were transfected and no significant difference between Lipofectamin or Turbofect was observed. However, more cells were transfected on early stages of SSC colony formation (7th days), especially when Turbofect was used (around 40 and 45% for Lipofectamin and Turbofect, respectively). Although the early stages SSCs were more suitable for transfection, but the formation of colonies were impaired on transfected cells.

Cloning Mice.

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos.

Comparative Stepwise Pattern of Reactive Oxygen Species Production during In Vitro Development of Fertilized and Nuclear Transferred Goat Embryos.

BackgroundA unique feature of embryo metabolism is production of reactive oxygen species (ROS). It is well established that during in vitro culture, ROS levels increase over normal ranges observed for embryos developed in vivo. This study evaluates and compares the stepwise pattern of ROS production during in vitro development of reconstructed goat embryos produced by zona-free method of somatic cell nuclear transfer (SCNT). Furthermore, the pattern of ROS production of SCNT embryos were compared with zona free embryos derived from in vitro fertilization (IVF).Materials and MethodsIn this experimental study, zona-free oocytes, SCNT and IVF embryos at different stages of in vitro development (2, 4, 8, 16-cells, morula, and blastocyst) were used for assessment of ROS production using 2, 7-dichloro dihydroflourescein diacetate (DCHFDA) probe and the result were presented as fold increase or decrease relative zona free oocytes.ResultsThe relative level of ROS compared to metaphase-II (MII) oocytes insignificantly decrease during early stages post embryo reconstitution and regained its value by 8-cell and morula stage and, significantly increase compared to MII oocytes by blastocyst stage.ConclusionThe pattern of ROS change in SCNT embryos is similar to zona free IVF derived embryos, except it decrease from two cell stage and regain its value at morula stage. The sudden rise in ROS at blastocyst stage, further emphasizes the special need of IVF and SCNT derived embryos during this stage of development.

SCNT Method and the Application for Patent Eligibility on Cloned Animals

Patents recognize economic right and are important for both individual and social economic benefit. Nonetheless, mere economic right does not eliminate the requirement for moral assessment when adjudicating intellectual property claims, especially in the case of claims associated with applications of biomedical technology [e.g., somatic cell nuclear transfer (SCNT) methods]. This is so for applications for patent in the case of live-born animal clones, as governed in the setting of the judicial system of the USA. Here recent federal court decisions in the USA are reviewed, and the ethical ambiguities of this judicial review are engaged in light of the current prohibitions on human reproductive cloning. It is concluded that the legal proscription of patents on animal clones bodes well for human accountability to present and future generations in the event of human reproductive cloning.

Editorial Vol.7(2)

Hello readers! Hope everyone is fine especially in this season where we often are prone to attacks of cold or flu. The holiday season is at our threshold, and we wish everyone to be in the best of health and happiness.This issue of the BJB is very interesting with topics stretching from Non Communicable Diseases to the ethical issues related to the habitation of the planet Mars, and proves how forward looking are our readers and authors.Mohammad Rashedul Islam et al discuss the application of the SARA method in a district in Bangladesh. The method is based on the principle of Service Availability and Readiness Assessment of WHO. The study in a district in Bangladesh found that about 62% of all the facilities were ready to provide general services like basic amenities with basic equipment, standard precautions for infection prevention, diagnostic capacity and essential medicines. However in case of Non-Communicable Diseases (NCD), only 40% of the health facilities had chronic respiratory disease and cardiovascular diseases diagnosis/management and only 32% had availability of diabetes diagnosis/management. Therefore, service availability and readiness of the health facilities to provide NCD related health services were not satisfactory. The authors emphasize that SARA which is a tool to monitor facilities to be ready for provision of services including NCD, should be used by all clinic managers.The very scientific and forward looking article titled “SCNT Method and the Application for Patent Eligibility on Cloned Animals” by Norman K. Swazo reviews US federal court decisions regarding applications for patent in the case of live-born animal clones. A decision issued from the United States Court of Appeals for the Federal Circuit established an earlier decision of the Patent Trial and Appeal Board that live-born animals cloned by the somatic cell nuclear transfer method (SCNT) like cattle, sheep, pigs, and goats are not patentable, so a scientist using the method is not eligible for a patent claim on the animals. The author feels that this decision has ethical implications related to the moral status of cloned animals. The author argues whether the end product of a SCNT (somatic cell nuclear transfer) application is same as an invention or only a revelation of nature at the cellular level. The author quotes Article 11 of Universal Declaration on the Human Genome and Human Rights UNDHGR “Practices which are contrary to human dignity, such as reproductive cloning of human beings, shall not be permitted.” He concludes that the judicial assessments in the USA deciding on the question of patenting of animal clones provide important barriers to human reproductive cloning research, safeguarding moral and legal commitment to human dignity. The article on Ethics in Public Health Research and Clinical Research by Muhammad Waseem Khanet al discusses on the need to continue to focus on ethical values in conducting any research. Tha authors stress the importance, either in public health research or clinical research, of the need to recognize an ethical standard that respects individual’s autonomy and community’s wellbeing. This can be achieved through collective collaboration for the protection of individual’s autonomy, dignity and wellbeing. Research ethics is a fundamental criterion to be complied with throughout a research study. The very interesting article on Planetary Protection for Mars: Time for Reconsideration by Svetoslav Alexandrov looks into the ethical questions beyond mother earth but which could be affected by people from our planet. In this manuscript the author discusses the ethics of the protection of hypothetical organisms on Mars in terms of planned manned space missions and subsequent colonization. The author discusses the possible problems related to a process called terraformation where the planet is converted to make it suitable for Earth life. He argues that terraformation will change the whole condition of the planet. The author quotes Carl Sagan, of The Planetary Society: “If there is life, then I believe we should do nothing to disturb that life. Mars then, belongs to the Martians, even if they are microbes” . I think we all agree with this brilliant recommendation.The article ‘We Still Need to Improve Our Research Writing Ethics’ by Sheikh Arslan Sehgal looks into the value of maintaining professional integrity of research work. Particularly with issues like plagiarism, ethics, authorship and data fabrication. He argues that the scientific community must obey the scientific ethical norms and rules. He feels that ethical problems must be resolved for the advancement of research in countries including Pakistan. The author recommends teaching students the international, institutional and professional standards regarding research and authorship issue. Thus from the beginning researchers will know the key components of ethics in conducting research and the end product will be research work of quality and acceptability.Dear Readers, we look forward to your articles, commentaries, observations on different issues which touch our lives and will help in broadening our knowledge and understanding of Bioethics.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cell nuclear transfer (SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout (KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear fibroblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cell colonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.

Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9934-1) contains supplementary material, which is available to authorized users.

The Complete Genome Sequences, Unique Mutational Spectra, and Developmental Potency of Adult Neurons Revealed by Cloning

Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor ∼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0-2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development.

Method for somatic cell nuclear transfer in zebrafish.

This chapter presents a detailed methodology for somatic cell nuclear transfer-cloning of zebrafish. We aim to place the reader in a virtual lab experience to assist acquisition of the technical skills required for reproducing the published protocol. All materials, including catalog numbers for reagents and techniques for their preparation, are provided. Our protocols describe laser inactivation of egg chromosomes, the transfer of a cell through the oocyte micropyle, and spontaneous activation of the reconstructed embryo. High-quality eggs are the key to cloning success, and Chinook salmon ovarian fluid is indispensable for keeping eggs arrested at the metaphase of meiosis II. This protocol continues to be refined by our laboratory. However, naive investigators should be able to apply it in its present form to generate cloned zebrafish.

Pluripotent stem cells and their use in hearing loss

Throughout its half a century of development, stem cell research has included two main fields: embryonic stem (ES) cell research and the reprogramming of body somatic cells. In the present review we focused on stem cell reprogramming and its relation with otolaryngology. The human body somatic cells are transformed into pluripotent cells by three basic methods: the somatic nuclear transfer method, the somatic cell fusion method (getting cellular pluripotent capacity in cellular reprogramming), and by transcription factors influencing the body somatic cells to generate reprogrammed induced pluripotent stem (iPS) cells. ES cells and iPS cells have pluripotency and differentiate into cells originating from the three germ layers; they are preferred for cellular treatment, drug development, and disease modeling research. Because of ethical restrictions in obtaining ES cells, iPS cells are an alternative pluripotent cell source and patient-specific autologous pluripotent cells are obtained by this method. Cellular treatment and regenerative medicine with pluripotent cells are currently developing and we aimed to raise awareness about this topic in our paper on using iPS cell technology in the biological treatment of hearing loss, which is an important area of research in otolaryngology.