PLUS Program Research Articles

82 Disruption of p53 in bovine somatic cells affected cloned embryonic development

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

Effect of Polystyrene-Sulfonate Surfactant on Reduction of Remaining Abrasives and Surface Roughness in Tungsten-Film Chemical-Mechanical-Polishing

The W-film chemical-mechanical-polishing (CMP) has been utilized for the formation of via-hole and metal wiring as an essential semiconductor process. In the W-film CMP, key performance parameters are high polishing rate, low surface roughness of W-film, and high selectivity between SiO2 and W-film CMP. Recently, the remaining nanoscale abrasives in sub-micron W lines has been seriously issued as the design rule has scaled down below 20 nm in memory devices, as shown in Fig.1. In addition, the surface roughness after W-film CMP has been important since it influences directly the metal contact resistance. Thus, in our study, we designed a novel W-film CMP slurry using the sulfonate functional group (i.e., polystyrene sulfonate (PSS)) and 10-nm-diameter ZrO2 abrasives to reduce remaining abrasive in the sub- micron W-lines and surface roughness after W CMP. Principally, a W CMP slurry was fabricated with nanoscale ZrO2, Fe based catalyst, corrosion inhibitor, pH stabilizer, and anionic surfactant (i.e., PSS). First, we investigated the dependency of the W-film polishing rate on the PSS concentration. The W-film polishing rate decreased from 126 to 53 Å/min when the concentration increased from 0 to 1.0 wt%, and then it increased from 53 to 100 Å/min when the concentration increased from 1.0 to 2.0 wt%, as shown in Fig. 2(a). This result indicates that the W-film polishing rate minimized at a specific concentration; i.e., 0.5 wt% in which mechanism will be presented in the conference. Second, the W-film surface roughness [root-mean-square (RMS) at 2 x 2-um scanning area] after CMP increased from 1.302 to 1.662 nm when the concentration increased from 0 to 0.5 wt%, and then it decreased from 1.662 to 1.289 nm when the concentration increased from 0.5 to 2.0 wt%, as shown in Fig.2(b). This result means that the surface roughness peaked at a specific concentration; i.e., 1.0 wt%. Finally, the area ratio of ZrO2 abrasives remained at sub-micron W lines after W-film CMP decreased from 95.7 to 2.4 % when the concentration from 0 to 0.5 wt%, and then it increased from 2.4 to 97.6 % when the concentration from 0.5 to 2.0 wt%, as shown in Fig. 3. This result imply that the area ratio of ZrO2 abrasives remained at sub-micron W lines after W-film CMP minimized at a specific concentration; i.e., 0.5 wt%. In our presentation, we will review the mechanism of polystyrene-sulfonate surfactant effect on tungsten-film chemical-mechanical-polishing properties improvement such as remaining abrasive in sub-micron W lines and surface roughness. Figure 1. SEM images of the remaining nanoscale abrasives in sub-micron W lines. Figure 2. Dependency of polishing rate (a) and surface roughness (b) as a function of PSS concentration Figure 2. Dependency of the area ratio of ZrO2 abrasives remained at sub-micron W lines after W-film CMP as a function of PSS concentration Acknowledgement This research was supported by the MOTIE(Ministry of Trade, Industry & Energy (10085643) and KSRC(Korea Semiconductor Research Consortium) support program for the development of the future semiconductor device. This investigation was financially supported by the Brain-Korea 21 PLUS Program in 2017 and Semiconductor Industry Collaborative Project between Hanyang University and SK Hynix Figure 1

Design of Perpendicular Magnetic-Tunneling-Junction Spin-Valve Including [Co/Pt]n Free Layer As Bulk Perpendicular Magnetic Anisotropy

Recently, dynamic random access memory (DRAM) has scaling limit because of its capacitor area. To replace DRAM, the next generation memory devices are being studied. Among them, perpendicular spin transfer torque magnetic random access memory (p-STT MRAM) is considered as a promising candidate because of easy scaling down, low power consumption, high read and write speed and non-volatility. p-STT MRAM has several challenges for commercialization. To keep a high retention time (~10 years), STT-MRAM should have thermal stability greater than 74. Although the thickness of free layer is increased with thermal stability, the thickness of free layer is limited to maintain perpendicular magnetic anisotropy (PMA). One of studies, double MgO based p-MTJ spin-valve structure is issued for its large volume because in case of double MgO based p-MTJ spin-valve, PMA of the free layer is originated from two i-PMA between the CoFeB free layer / MgO tunneling barrier and CoFeB free layer / MgO capping layer. The i-PMA induced from the two interfaces increases the total volume of the magnetic free layer which increases the thermal stability, but it is still limited by the thickness of the individual CoFeB free layers. In our study, therefore, we propose a new structure using interfacial Co2Fe6B2 and [Co/Pt]n multi-layer as bulk PMA to achieve higher thermal stability than that of double MgO based p-MTJ spin-valve structure. The dependence of the magnetic moment and thermal stability on the number of [Co/Pt]n multi-layers was estimated by using vibrating sampling magnetometer (VSM) as shown in Fig.1 b. Magnetic moment t~180μemu/cm2) of i-PMA+[Co/Pt]2 multi-layers is similar to that(~180μemu/cm2) of double MgO based p-MTJ spin-valve structure. Also, magnetic moment (t~366μemu/cm2) of i-PMA+ [Co/Pt]5 multi-layers is about two times more than that of double MgO based p-MTJ spin-valve structure and thermal stability (~181) is two times higher than that (~74) of the double MgO based p-MTJ spin-valve structure. The thermal stability can be modulated by simply changing the number of [Co/Pt]n multi-layers. Obviously, these results show that i-PMA + [Co/Pt]n multilayer as bulk PMA based p-MTJ spin-valve can achieve a high thermal stability. In our presentation, we report the different characteristics of the p-MTJ spin-valve with [Co/Pt]n multi-layers by examining the static magnetization behavior, TMR ratio and MgO crystallinity. In addition, we present how these properties influence mechanism of p-MTJ spin-valve. Acknowledgements This work was supported by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2017R1A2A1A05001285) and the Brain Korea 21 PLUS Program in 2014. Figure 1

Design of Double Pinned Perpendicular Magnetic-Tunnel-Junction Spin-Valve Exhibiting Multiple Resistance States

Perpendicular spin-transfer-torque magnetic random access memory (p-STT MRAM) cells consist of a perpendicular magnetic tunnel junction and a selective device has been researched intensively because of the possibility of overcoming the scaling limitations of the current dynamic random access memory (DRAM) below the 10-nm design rule [1]. p-STT-MRAM requires high tunneling magnetoresistance (TMR) ratio greater than 150%, sufficient thermal stability (Δ=KuV/kBT) above 75 for a ten-year retention-time, and low switching current (J C0 ~ 1 MA/cm2) must be achieved for low power consumption [2-3]. However, the thermal stability is hard to maintain when scaling down in 10-nm scale of the p-STT MRAM cells for terabit integration. It requires high interfacial anisotropy K i to maintain the thermal stability requirement for 10-year retention time [4]. In this study, we designed a p-MTJ spin-valve with a double pinned structure to realize two-bit operation to secure a larger scaling limit (see Fig. 1(a)). We investigated the dependency of the number of [Co/Pt]n multilayers on the coupling strength to the pinned layers in order to control the spin-electron direction of the pinned layers. The total number of [Co/Pt]n SyAF multilayers ferro-coupled to the bottom pinned layer was reduced from 9 to 3 layers to reduce the roughness of the MgO tunneling barrier to increase the TMR ratio. Also, the number of [Co/Pt]n of the top upper SyAF multilayers was modulated so that the coercive field (H c) required to switch the spin-electron direction of the top pinned layer increased from about 0.3 kOe to 1 kOe which is sufficient to ensure the memory margin for two-bit operation, as shown in Fig. 1(b). Then, we investigated the multiple resistance states with the R-H loop of the double pinned p-MTJ spin-valve, as shown in Fig. 1(c). It showed four different resistance states depending on the spin-electron direction of the free layers and pinned layers as shown in Fig. 1(d) with a maximum TMR ratio of 166%. This implies that the double pinned p-MTJ spin-valve may be suitable for terabit integration compared to that of the single-bit operating p-STT MRAM memory cells. Acknowledgements This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2017R1A2A1A05001285) and Brain Korea 21 PLUS Program in 2014.

Effect of Misfit Dislocations in Junctions and Base Thickness on Latch-up Characteristics in Two-Terminal Vertical Thyristor-Based Capacitorless Memory

Conventional dynamic random access memory (DRAM) has been facing a severe challenge to scale down to 10 nm size. The cell capacitor should be able to store sufficient charges of 25~30 fF/cell. In order to meet this requirement, high aspect ratio of cell capacitor is inevitable, resulting in capacitors leaning into each other. To overcome this issue coming from capacitor, the vertical thyristor-based two-terminal capacitorless memory was proposed as a promising candidate to replace current DRAM, which consists of p++-anode / n+-base / p+-base / n++-cathode vertical structure using conventional Si technology. The two-terminal vertical thyristor-based capacitorless memory cell having p++-anode / n+-base / p+-base / n++-cathode vertical structure is easy to construct cross-point memory array cells, as shown in Figure 1. Furthermore, Two-terminal vertical thyristor-based capacitorless memory array cells can be operated without selector since sneak current is suppressed by latch-down process. In our study, three types of two-terminal vertical thyristor-based capacitorless memory cells consist of top-p++-anode / n+-base / p+-base / bottom-n++-cathode with 80-nm-thick base region (PNPN80), top-p++-anode / n+-base / p+-base / bottom-n++-cathode with 160-nm-thick base region (PNPN160), and top-n++-cathode / p+-base / n+-base / bottom-p++-anode with 160-nm-thick base region (NPNP160) were fabricated, individually. The dopant concentration profile of each device was analyzed by secondary ion mass spectroscopy (SIMS), as shown in Figure 2. By correlating HR TEM images with SIMS analyses, misfit dislocations were observed at the depth where dopant pile-up was found, as shown in Figure 3. Figure 4 (a), (b) and (c) show I-V measurements of PNPN80, PNPN160 and NPNP160, respectively. The difference of latch-up voltages between Figure 4 (b) and (c) can be explained by carrier life-time difference. NPNP160 had less misfit dislocations compared to PNPN160, resulting in longer excess carrier life-time, since dislocations act as recombination centers. Thus, NPNP160 needed smaller anode voltage to induce sufficient excess carriers in base regions, which had smaller latch-up voltage than PNPN160. In addition, the difference of latch-up voltages between Figure 4 (a) and (b) resulting from base thickness difference can be explained by electric field. The electric field of PNPN160 is weaker than that of PNPN80 in base regions when equivalent anode voltage is applied. Therefore, PNPN160 needed larger anode voltage to lower the injection barrier of excess carriers. Besides, the effect of misfit dislocations and base dopant concentration on memory margin and off-state leakage current will be discussed. Finally, the memory cell pulse operations will be exhibited as well. Acknowledgment * This research was supported by Brain Korea 21 PLUS Program in 2018, the MOTIE (Ministry of Trade, Industry & Energy 10069063) and KSRC (Korea Semiconductor Research Consortium) support program for the development of the future semiconductor device. Figure 1

Empower, rather than rescue, students on the path to college success

Traci Tuttle, Ed.D., is the director of the PLUS program and the Disability Education Office at Muskingum University. The PLUS program is a comprehensive support services program for students with learning differences.

Türkçe ve Sosyal Bilimler Eğitimi Bölümündeki Öğretmen Adaylarının “Kitap” Kavramı Üzerine Algıları

The purpose of this study is to determine the perceptions of teacher candidates of Turkish and Social Sciences Education on the concept of "book" through metaphors. The study group consists of 330 students who study in the first and last classes of Turkish and Social Sciences Education departments in the second semester of 2016-2017 academic year. The data were obtained through the forms in which the personal information was included in the first part and the completed form was given in the second part "book for me ... because ...". The 330 data obtained were analyzed by content analysis technique via Nvivo 11 PLUS program and 9 metaphors determined to be weak structure were extracted without working. The remaining 321 metaphors were compiled to form a metaphor list, grouped by various conceptual themes, and 13 conceptual categories were created. According to the metaphor numbers in these 13 conceptual categories; journey (111), source of information and entertainment (65), friend (44), treasure (22), awareness (22), nature (17), object (11), requirement (11), life (7), insignificant (4), feeling (3), purpose (2) and miracle (2).

Mentoring for medical students : Description and evaluation of adifferentiated mentoring program at amedical school

In recent years, mentoring programs for medical students have been increasingly implemented in medical schools in Germany. This article describes avoluntary mentoring program for students, which is differentiated into acommon mentoring program (AP), amentoring program for excellent students (EP) and amentoring plus program (PP) for students with academic difficulties, and its evaluation.The evaluation is based on annual evaluation surveys among the participating mentees, who evaluate the mentoring relationship as well as the impact on their studies.The three student groups differ significantly with regard to sociodemographic variables. The satisfaction with the mentoring program is generally high: 84-94% are mentored by their preferred mentor, the majority assesses the conversational atmosphere as open (78-91%), and 89-98% would recommend participation in the program to others. The programs differ with regard to specific aspects. While PP mentees report the highest ratings on the mentor's accessibility, trustworthiness and feedback, EP mentees report the highest ratings on the mentor's competence as well as increased identification with the faculty and early career promotion through the mentoring program.The different assessments of the three programs as well as the generally high acceptance and satisfaction ratings indicate that such adifferentiated mentoring program provides aframework of individual support, which is highly appreciated by most students.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

204 Blood Analysis of Cas9-Expressing Cattle

The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm3), neutrophils (4590 and 3870/mm3), lymphocytes (4020 and 5910/mm3), red blood cells (732,000 and 798,000/mm3), hemoglobin (9.5 and 10.2 g dL−1), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm3), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL−1), and creatinine (1.3 and 1.0 mg dL−1) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization. This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).

27 Quisinostat, a Potent Histone Deacetylase Inhibitor, Regulates the Expression of Pluripotency- and Reprogramming-Related Genes on Somatic Cell Nuclear Transferred Porcine Embryos

The low efficiency of somatic cell nuclear transfer (SCNT) has been attributed mostly to inefficient epigenetic reprogramming. Recently, various histone deacetylase inhibitors (HDACi) were used to improve developmental competence of SCNT embryos in several species. However, limited information is available on the effects of quisinostat (JNJ-26481585, JNJ), a second-generation HDACi with high cellular potency towards Class I and II histone deacetylases. Based on our previous study, among various concentrations, treatment with 100 nM JNJ could improve embryo development into blastocysts compared with the control (23.50 ± 1.30 v. 13.97 ± 1.37; P < 0.05). Thus, in the present study, treatment with 100 nM JNJ was used for further investigation into the relative expression of genes related to pluripotency and reprogramming in order to assess the quality of pre-implantation embryos cultured in media with JNJ using quantitative real-time PCR. Porcine fibroblasts isolated from kidney of adult pigs from passage 6 to 8 were used as nuclear donor cells for SCNT. After SCNT, embryos were cultured with or without 100 nM JNJ during the first 24 h of in vitro culture, and blastocysts from each experimental group were collected and kept at –80°C until analysis. Total RNAs were extracted, and transcribed into cDNA before amplification. Then, the relative expression of development-related (Oct4, Sox2, and Nanog), histone acetylation-related (HDAC1, HDAC2, and HDAC3) and DNA methylation-related (DNMT1, DNMT3a, and DNMT3b) genes between the control and 100 nM JNJ groups were compared. All experiments were repeated 4 times and results were analysed by independent t-test using SPSS 17.0K (SPSS Inc., Chicago, IL, USA). Treatment with 100 nM JNJ showed significant increases in the expression Oct4, Sox2, and Nanog compared with the control (P < 0.05). Moreover, there was significantly lower expression of HDAC1, HDAC2, HDAC3, DNMT1, DNMT3a, and DNMT3b in the 100 nM JNJ treatment than in the control (P < 0.05). These expression results moderately illustrated more active transcriptional factors, stable maintenance of embryonic pluripotency, and lesser activity of histone acetylation and DNA methylation enzymes, enhancing the blastocyst formation rate in the treatment group. In conclusion, we suggest that improvement of the in vitro developmental competence of porcine SCNT embryos might be related to positive regulations of JNJ on the expression levels of genes related to pluripotency and reprogramming. This study was supported by the NRF (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

Enhancement of Switching Property of Perpendicular- Magnetic Tunnel Junctions Using Single MgO Based Co2Fe6B2 Double Free Layer Structure

Recently, perpendicular-spin-transfer torque magnetic random access memory (p-STT MRAM) has received considerable attention as the next-generation memory referred from its pre-eminent characteristics. It has high speed writing and reading property like dynamic random access memory (DRAM) with an additional property of non-volatility. Furthermore, it also has stable endurance (> 1014), less electric energy consumption to maintain its data (~1 pJ/bit) and high scalability. Because of these properties, p-STT MRAM has been considered to be the universal memory that can replace other memories in the market. However, it is difficult to be commercialized due to many critical issues of device parameters i.e., the tunneling magnetoresistance (TMR) ratio, thermal stability (Δ), and switching current density (JC). In particular, theses three device parameters should be achieved at 400oC temperature of the back-end of line (BEOL) to be manufactured into the market. To gain a reputation as a non-volatile memory at the market, p-STT MRAM needed low switching current density for low-power operating device. In our research, we investigated new MTJ structure using single MgO based double Co2Fe6B2free layers separated by a W spacer layer (See Fig.1) for low current switching. Samples were made by utilizing 12-inch multi-chamber magnetron sputtering system and magnetic vacuum oven. After sputtering process, samples were ex-situ annealed at 350 or 400oC under a perpendicular magnetic field of 3T for 30 minutes. Figure 2 shows the switching voltage of anti-parallel state to parallel state (AP-P) and parallel state to anti-parallel state (P-AP) on I-V measurement. The switching voltage of AP-P and P-AP at newly double free layer decreased about 3.8 and 5.2% respectively, compared with the conventional free layer. This means that upper Co2Fe6B2 free layer had an influence for the lower Co2Fe6B2 free layer, therefor the lower Co2Fe6B2free-layer easily switched to perpendicular direction by in-plane magnetic anisotropy (IMA) magnetic resonance coupling. In our presentation, we will present the switching property of Co2Fe6B2 double free layer for p-MTJs and review the switching mechanism for IMA magnetic resonance coupling by using IV measurement. In addition, we review the magnetic properties, atomic composition and TMR ratio, by using vibrating-sampling-magnetometer (VSM), Secondary-Ion-Mass-Spectrometry (SIMS), current-in–plane-tunneling (CIPT) measurement respectively. * This work was supported by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2014R1A2A1A01006474) and the Brain Korea 21 PLUS Program in 2014. Figure 1

Double MgO Based Perpendicular Magnetic-Tunnel-Junction Spin-Valve without Upper Synthetic-Anti-Ferromagnetic Multi-Layers

Recently, the semiconductor memory device has faced the difficulties of scaling down, low power consumption and high retention time. In particular, dynamic random access memory (DRAM) has been struggling with scaling limit because of reducing capacitor area. In addition, alternative memories have been studied intensively to replace DRAM. Among them, perpendicular spin transfer torque magnetic random access memory (STT-MRAM) is considered as a strong candidate because of the small scaling down, low power consumption, high read and write speed and non-volatility. STT-MRAM has the structure of 1 transistor and 1 resistance (1T-1R) called magnetic tunnel junction (MTJ). Resistance difference between parallel and antiparallel states in MTJ indicates memory sensing margin. The large difference of tunneling magneto-resistance (TMR) is required for a good memory operation. To obtain the high TMR ratio, not only perfect switching of the free layer but also hard pinning of the pinned layer is required. It is known that pinning the pinned layer is due to the exchange coupling induced by a synthetic anti-ferro-magnetic (SyAF) layer based on Ruderman Kittel Kasuya Yosida (RKKY) interaction. In our study, therefore, we investigated how to optimize the SyAF layer structure for the proper exchange coupling. MgO tunnel barrier should be b.c.c (100) crystallinity because it acts as an electrons filter which determined whether spin direction between free and pinned layers is aligned parallel or not. In particular, we propose double MgO based perpendicular magnetic-tunnel-junction spin-valve without upper synthetic-anti-ferromagnetic Multi-layers in order to obtain high TMR ratio. In addition, we investigated the different characteristics of the p-MTJ spin-valve with and without upper SyAF multi-layers. For the experiment, two structures, double MgO based p-MTJ spin-valve with and without upper SyAF multi-layers, were fabricated on a 12-inch-diameter wafer deposited with SiO2/W/TiN films in a 12-inch multi-chamber sputtering system under a high vacuum of less than 1ⅹ10-8 torr (without breaking the vacuum; see Figs. 1a and b). The thicknesses of the tungsten (W) were varied ranging from 0.18 to 0.48 nm. All samples were subject to ex-situ annealing at 350oC for 30 min under a perpendicular magnetic field of 3 Tesla. The dependence of the TMR ratio on the thickness of W bridge layer was estimated by using current-in-plane tunneling (CIPT) at room temperature as shown in Fig.1c. It shows that a TMR ratio of p-MTJ spin-valve with upper SyAF multi-layers slightly increases at the bridge layer thickness, t w up to around 0.3 nm. Then it abruptly decreases when the thickness was increased from 0.36 to 0.5 nm. On the other hand, the TMR ratio of p-MTJ spin-valve without upper SyAF multi-layer increases at the t w up to around 0.24 m. Then, the TMR ratio abruptly decreases when the thickness was increased from 0.36 to 0.4 nm. And maximum TMR ratio of p-MTJ spin-valve with upper SyAF multi-layers is 156 % while, that of p-MTJ spin-valve without upper SyAF multi-layers is 180 %. Obviously, the TMR ratio of p-MTJ spin-valve without upper SyAF multi-layers is higher 24 % than p-MTJ spin-valve wih upper SyAF multi-layers. In our presentation, we report the different characteristics of the p-MTJ spin-valve with and without upper SyAF multi-layers by examining the static magnetization behavior, MgO crystallinity, and I-V characteristic curve. In addition, we present how these properties influence the TMR ratio and mechanism of p-MTJ spin-valve. * This work was supported by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2014R1A2A1A01006474) and the Brain Korea 21 PLUS Program in 2014. Figure 1

205 PRODUCTION OF Cas9-EXPRESSING CATTLE USING DNA TRANSPOSON

A genome-editing technology, CRISPR/Cas9 system is proved to be a powerful tool for knockout and knock-in in various species. When 2 components [Cas9 and single guide (sg) RNA] are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for every gene editing based on the CRISPR/Cas9 system, some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals. Accordingly, if the Cas9-expressing cattle is born, it would be broadly used for gene editing in cattle. For this study, the Cas9 and RFP genes were cloned into the PiggyBac transposon system. PiggyBac-Cas9-RFP and transposase were microinjected into 1436 IVF embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounted for 14.1% of total formed blastocysts. Five blastocysts were selected and transferred into 5 recipient cow (1 embryo per recipient). After gestation periods, 4 transgenic cattle were delivered without any veterinary assistance. From transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNA in DNA form for various genes such as PRNP, RB1, and BLG were transfected with 2 μg of sgRNA per 5 × 105 cells using electroporation. As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1 assay. The data demonstrated that, for the first time, transgenic cattle with Cas9 expression were born, grown up to date (age = 5 months) and will be a valuable resource for genome editing in cattle. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

204 POTENTIAL OF GREEN FLUORESCENT PROTEIN LOCUS FOR GENE EDITING IN DNA TRANSPOSON-PRODUCED TRANSGENIC CATTLE

Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated that transgenic cattle via transposon are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the target region for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on disease resistance and food production. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

The effectiveness of the peer-delivered Thinking Healthy PLUS (THPP+) Program for maternal depression and child socioemotional development in Pakistan: study protocol for a randomized controlled trial

The effectiveness of the peer-delivered Thinking Healthy PLUS (THPP+) Program for maternal depression and child socioemotional development in Pakistan: study protocol for a randomized controlled trial

Thermally Stable Perpendicular-Magnetic Tunneling Junction Spin-Valve Implementing W Bridge, Space and Cap-Layer

Perpendicular spin-transfer-torque magnetic random access memory (p-STT MRAM) has been intensively researched because of the possibility of it overcoming the scaling limitations of current dynamic random access memory (DRAM) below the 10-nm design rule1. p-STT MRAM cells consist of a selective device and a perpendicular magnetic tunneling junction (p-MTJ) spin-valve. Moreover, a lot of research has gone into improving three device parameters of these devices, i.e., the tunneling magnetoresistance (TMR) ratio, thermal stability (Δ), and switching current (JC ), at the back end of line (BEOL) temperature of 400oC3. Note that the BEOL temperature of 400oC is required in order to integrate memory cells3. Tantalum (Ta) has been used as a bridge and spacer material to ferro-couple two ferro-magnetic layers. However, so far, it has proven extremely difficult achieve a high TMR ratio in a MgO based p-MTJ spin-valve using a thickness Ta spacer at the BEOL temperature of 400oC4. In our experiment, we tried a new spacer material with a body-centered-cubic (bcc) crystal structure (i.e., tungsten). We investigated the dependency of the TMR ratio on the thickness and type of material (tantalum or tungsten) of spacers in double MgO-based p-MTJ spin-valves with a top free Co2Fe6B2 layer. Two types of double MgO based p-MTJ spin-valves with a [Co/Pt]n-based SyAF layer were fabricated on a 12-inch-diameter wafer deposited with SiO2/W/TiN films in a 12-inch multi-chamber sputtering system under a high vacuum of less than 1×10−8 torr (without breaking the vacuum; see Figs. 1a and b). The thicknesses of the Ta and W spacers were varied between 0.2 and 0.7 nm. All samples were subject to ex-situ annealing at 400oC for 30 min under a perpendicular magnetic field of 3 Tesla. The dependence of the TMR ratio on the thickness and type of material of the spacer was estimated by using current-in-plane tunneling (CIPT) at room temperature, as shown in Fig. 1c. For the Ta spacers, the TMR ratio significantly increased from ~38 to ~95% as the spacer thickness was increased from 0.20 to 0.35 nm. It remained about 98% for spacer thicknesses ranging from 0.40 to 0.53 nm. It decreased from ~90 to ~42% as the spacer thickness was further increased from 0.58 to 0.70 nm. Thus, the TMR ratio (~98%) peaked in a certain spacer thickness region (0.40~0.53 nm) for Ta. Next, regarding the samples made with W spacers, the TMR ratio significantly increased from ~85 to ~131% as the spacer thickness was increased from 0.20 to 0.30 nm. It remained at ~134% as the spacer thickness was further increased from 0.40 to 0.53 nm. The ratio slightly decreased from ~134 to ~111% for larger thicknesses ranging from 0.58 to 0.70 nm. Thus, the TMR ratio (~134.0%) also peaked in a specific spacer thickness region (0.40~0.53 nm) for W. Comparing the TMR ratios in the cases of the Ta spacer and the W spacer obviously indicates that the use of 0.20 and 0.70 nm-thick W spacers resulted in least 35% higher TMR ratios in the comparison with the use of Ta spacers. In particular, both the thinnest (0.20 nm) and thickest (0.70 nm) W spacers yielded much higher TMR ratios (~85 and ~111%) in comparison with the corresponding Ta spacers (~38 and ~42%). In our presentation, we will report the mechanism by which thickness and material of the spacer influence the TMR ratio was revealed by examining the static magnetization behavior, (100) bcc crystallinity, and depth profile of the atomic composition of the spin-valves. In addition, we will present the result of effects on W bridge/cap-layer compare with Ta bridge/cap-layer in Double MgO based p-MTJ spin-valve. * This work was supported by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2014R1A2A1A01006474) and the Brain Korea 21 PLUS Program in 2014. Reference [1] K. C. Chun et al., IEEE J. Solid-st. Circ. 48, 598 (2013) [2] K. Yamane et al., IEEE T. Magn. 49, 4335 (2013). [3] L. Thomas et al., J. Appl. Phys. 115, 172615 (2014) [4] J. Swerts et al., Appl. Phys. Lett. 106, 262407 (2015) Figure 1

A Clustered Randomized Controlled Trial of the Positive Prevention PLUS Adolescent Pregnancy Prevention Program.

To determine the impact of Positive Prevention PLUS, a school-based adolescent pregnancy prevention program on delaying sexual intercourse, birth control use, and pregnancy. I randomly assigned a diverse sample of ninth grade students in 21 suburban public high schools in California into treatment (n = 2483) and control (n = 1784) groups that participated in a clustered randomized controlled trial. Between October 2013 and May 2014, participants completed baseline and 6-month follow-up surveys regarding sexual behavior and pregnancy. Participants in the treatment group were offered Positive Prevention PLUS, an 11-lesson adolescent pregnancy prevention program. The program had statistically significant impacts on delaying sexual intercourse and increasing the use of birth control. However, I detected no program effect on pregnancy rates at 6-month follow-up. The Positive Prevention PLUS program demonstrated positive impacts on adolescent sexual behavior. This suggests that programs that focus on having students practice risk reduction skills may delay sexual activity and increase birth control use.

(Invited) New Design of Double MgO-Based p-Mtj Spin-Valves with Top Co2Fe6B2 Free Layer Performain Highly Increased TMR Ratio and Thermal Stability

Perpendicular spin-transfer-torque magnetic-random-access-memory (p-STT MRAM) has intensively been researched as a promising memory cell to overcome a physical scaling limit of less than 20 nm in current dynamic-random-access-memory (DRAM) because of its fast write time (~10 ns), non-volatile memory operation, and extremely low power consumption. For realizing terra-bit-level integration of p-STT-MRAM cells fabricated with a selective transistor and a perpendicular-magnetic-tunnel-junction (p-MTJ) spin-valve, the p-MTJ spin-valves with a bottom CoFeB free layer using a Ta bridging and capping layer have been generally and widely researched. They need to achieve a high tunneling-magneto-resistance (TMR) ratio (>150%), thermal stability (Δ) (>74), and switching current (Jc) (~1x102 MA/cm2) at the back end of line (BEOL) temperature of 400oC. It has been reported that a Ta bridging and capping layer would be a good absorption layer for Boron (B) to enhance i-PMA characteristic but Ta atoms easily diffuse into the MgO tunneling barrier during ex- situ annealing temperature (or a thermal budget at BEOL)1,2. Thus, the p-MTJ spin-valves with a bottom CoFeB free layer using a tantalum (Ta: body-centered-cubic structure) bridging and capping layer have extreme difficulty achieving the TMR ratio of > 100% at the BEOL of 400 oC since the TMR ratio decreases rapidly as ex- situ annealing temperature (or a BEOL temperature) increases. Recently, the p-MTJ spin-valves with a top CoFeB free layer using a Ta bridging and capping layer have been intensively studied to obtain a higher TMR ratio at a higher ex- situ annealing temperature (or a BEOL temperature) since it would minimize the diffusion of platinum (Pt) atoms into the MgO tunneling barrier3-5. However, they confront a significant diffusion of Ta atoms into the MgO tunneling barrier at ex- situ annealing (or BEOL) of 400oC, so the b.c.c crystallinity of the MgO tunneling barrier is abruptly degraded, thereby rapidly decreasing the TMR ratio of the p-MTJ spin-valves. An innovative solution proposed in this paper is a new design of a p-MTJ spin-valve with a top Co2Fe6B2 free layer using a W bridging with sub-nanoscale thickness (~0.24 nm) and a tungsten (W: body-centered-cubic structure) capping layer with nanoscale thickness (~4.0 nm). The proposed design demonstrated no diffusion of Ta atoms into the MgO tunneling barrier and minimized the diffusion of Fe atoms at both Co2Fe6B2 free and pinned layer at an ex-situ annealing of 400oC. Therefore, b.c.c crystallinity degradation of the MgO tunneling barrier could be prevented, and high PMA magnetic moments at both Co2Fe6B2 free and pinned layers could be achieved. Thus, the p-MTJ spin-valve with a Co2Fe6B2 free layer using a W bridging and capping layer with nanoscale thickness (~4.0 nm) achieved the TMR ratio of ~143% at ex- situ annealing of 400oC, as shown Fig. 1. Furthermore, as a way to achieve a TMR ratio of >150% at 400°C, in this paper, we present the design of a new double MgO-based p-MTJ spin-valve with a top Co2Fe6B2 free layer by inserting a nanoscale Fe diffusion barrier (~0.30 nm: only two Fe atomic layers thick) between the W capping layer (~ 4.0 nm in thickness) and MgO capping layer (~1.0 nm in thickness). This new p-MTJ spin-valve had a TMR ratio of ~154% and Δ of 90 in ex-situ magnetic annealing at 400°C, as shown Fig. 2. * This work was supported by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2014R1A2A1A01006474) and the Brain Korea 21 PLUS Program in 2014. Reference [1] J. Appl. Phys. 2009, 106, 023920 [2] Acta Materialia 2015, 87, 259 [3] IEDM 2015, pp. 26. 2 [4] IEDM 2015, pp. 26. 3 [5] IEDM 2015, pp. 26. 4 Fig. 1. (a) p-MTJ spin-valve structure using a Ta bridging and capping layer, (b) p-MTJ spin-valve structure using a W bridging and capping layer, and (c) dependency of the TMR ratio on bridging and capping layer thickness for both p-MTJ spin-valves using Ta and W bridging and capping layers. Fig. 2. Dependence of TMR ratio on nanoscale thickness of Fe diffusion barrier for double MgO-based p-MTJ spin-valves with a top Co2Fe6B2 free layer. Figure 1

Breast-Cancer-Stem-Cell Sensor Using Igzo Nomfet Implemented with Au Nano-Particles

Recently, many researchers have studied circulating-cancer-stem-cell (CCSC) to pre-recognize the presence of a cancer. Since, it has been that cancer cells include negative or positive charges, several electronic sensors have been attractively proposed. In our study, we designed an In-Ga-Zn-oxide (IGZO) based FET implemented with Au nano-particles on IGZO channel, as shown in Fig. 1(a). The average size and density of the Au nano-particles on the channel were 12.05 nm and 2.66×1011 cm-2, respectively. In particular, this FET has a unique time-dependent current characteristic such as depressing at a positive gate bias or facilitating at a negative bias, which determines whether or not the cancers present a fixed charge or mobile charges. In our experiment, breast-cancer-stem-cell (BCSC) connected with the cancer-anti-body (CD44) was dropped on the IGZO FET with Au nano-particles where a linker (8-Mercaptooctanoic acid) was attached on Au nano-particles, as shown in Fig. 1(b). The linkers were well uniformly absorbed and attached on Au nano-particles, confirmed by FT-IR, as shown in Fig. 2. First of all, we investigated the dependency of threshold voltage on the presence of Au nano-particles, linkers, and breast-cancer-stem-cells with anti-bodies shown in Fig.3. The threshold voltage of the IGZO FET without Au nano-particles was +2.4 V. The implementation of Au-nano-particles on the IGZO channel increased from +2.4 to +12.0 V because of the presence of positive fixed charges at the interface between the IGZO channel and Au nano-particles. The attachment of the linker on Au nano-particles decreased the threshold voltage from +12.0 V to +6.9 V since the functional group of the linker was carboxyl. Two kinds of breast-cancer-stem-cells were dropped on the linker connected with Au-nano-particles; i.e., the cells without or with anti-body. The cells without anti-body did not shift threshold while the cells with anti-body reduced -4.3 V where the concentration of BCSC was 8000ea/10ul. The threshold-voltage shift amount was well correlated with the concentration of BCSC, as shown in Fig. 4. The threshold-voltage shift amount increased linearly with the concentration of BCSC, having the slope of the threshold-voltage shift amount of 0.44V/(kea/ul). In addition, we confirmed that BCSC showed a negative fixed charge rather than mobile charge by testing time dependent drain-current characteristic. In our presentation, we will review the detailed mechanism by which BCSCs shift threshold voltage and show a negative fixed charge characteristic. Reference [1] F. Alibart et al. Adv. Funct. Mater. 20 330. (2010)[2] K.-C. Kwon et al. Appl. Phys. Exp. 6, 067001 (2013).[3] XiuHua Zhang et al. Sensors, 3, 61-68 (2003) Acknowledgment This research was supported by the Converging Research Center Program funded by the Ministry of Science, ICT & Future Planning (Project No. 2015054348) and supported by Brain Korea 21 PLUS Program in 2014 Figure 1