Pyrethroid resistance in cockroach populations has been a public health challenge since the 1950s. The pyrethroid resistance in the German cockroach, Blattella germanica, is primarily attributed to knockdown resistance (kdr) mutations (E434K, C764R, and L993F) in the voltage-sensitive sodium channel gene (vssc). In this study, the pyrethroid resistance state of the German cockroach in the Republic of Korea (ROK) was assessed by analyzing the frequencies of kdr mutations using one-step PCR with total RNA. The results revealed that among the 25 populations examined, 14 populations exhibited the L993F kdr mutation, while no other mutations were detected. Since other cockroach species are also commonly found in human dwellings in ROK, the vssc genes were cloned from four other species, including Blattella nipponica, Periplaneta americana, Periplaneta japonica, and Periplaneta fuliginosa. Based on the genomic DNA (gDNA) sequences obtained from the vssc cloning, primer sets were designed to amplify the vssc fragment spanning the L993F mutation for each species and used to monitor the development of pyrethroid resistance in cockroach populations in the ROK. The study will facilitate the implementation of a nationwide monitoring program to assess cockroach resistance and select suitable alternatives.

Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

Paired box protein, PAX7, is a key molecule for the specification, maintenance and skeletal muscle regeneration of muscle satellite cells. In this study, we identified and characterized the cDNA and amino acid sequences of PAX7 from black sea bream (Acanthopagrus schlegelii ) via molecular cloning and sequence analysis. A. schlegelii PAX7 cDNA was comprised of 1,524 bp encoding 507 amino acids and multiple sequence alignment analysis of the translated amino acids showed that it contained three domains including paired DNA-binding domain, homeobox domain and OAR domain which were well conserved across various animal species investigated. Pairwise Sequence Alignment indicated that A. schlegelii PAX7 had the same amino acid sequences with that of yellowfin seabream (A. latus ) and 99.8% identity and similarity with that of gilt-head bream (Sparus aurata ). Molecular phylogenetic analysis confirmed that A. schlegelii PAX7 formed a monophyletic group with those of teleost and most closely related with those of the fish that belong to Sparidae family including A. latus and S. aurata . In the investigation of its tissue specific mRNA expression, the expression was specifically identified in skeletal muscle tissue and a weak expression was also shown in gonad tissue. The cultured cells derived from skeletal muscle tissues expressed PAX7 mRNA at early passage but the expression was not observed after several times of subculture.

This study investigated the effect of variation in the number of somaticcell- cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100- 150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

A γ-aminobutyric acid (GABA) producing microorganism was isolated from Sun-Tae Jeotgal, a Korean traditional fermented seafood. Two thousand presumptive lactic acid bacteria (LAB) isolates were screened for GABA production by thin layer chromatography. One isolate, T118, produced GABA profusely, and identified as Lactobacillus brevis. Growth of Lb. brevis T118 was examined during 120 h cultivation in MRS broth under different conditions. Lb. brevis T118 grew well at 30-37℃, initial pH of 4-7, and up to 5% NaCl (w/v). A gene, gadB, encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was cloned and gadC located immediately upstream of gadB, indicating gadCB operon structure. The operon structure was confirmed by reverse transcription (RT)-PCR. gadB was overexpressed in Escherichia coli BL21 (DE3) and recombinant GAD was purified. The size of recombinant GAD was 54.4 kDa by SDS-PAGE, which matched well with the calculated size from the nucleotide sequence.

This experiment was conducted to analyse the effects of flavone supplementation on the preimplantation development of in-vitro produced porcine embryos. During in-vitro development, immature oocytes and early embryos were exposed to different concentrations of flavone (0, 1μM, 25μM, 50 μM, and 100 μM respectively). Results showed that 100 μM of flavone significantly reduced the intracellular ROS levels of oocytes accompanied with a significant rise in GSH level. In parthenogenesis, no significant change was observed in the cleavage rates whether flavone was supplemented in IVM or IVC media. In IVM supplemented group, the blastocyst development rate was significantly enhanced by 1 μM concentration than other groups (51.5% vs. 41.3%, 44.0%, 36.3%, 31.7%; P<0.05) respectively. However, in IVC group 1 μM concentration significantly improved the blastocysts production than 50 μM and control groups (50.0% vs. 40.5%, 38.0%; P<0.05) respectively. Following nuclear transfer, the cleavage rate of IVM group was significantly more in 1 μM than 50 μM and 100 μM groups (92.9% vs. 89.7%, 87.8%; P<0.05), followed by similar pattern of cloned blastocysts production being significantly higher in 1 μM group than 50 μM, 100 μM and control groups (16.8% vs. 9.0%, 7.1%, 12.8%; P<0.05) respectively. In IVC group, 1 μM concentration resulted in significantly higher cleavage rate than 25 μM and 50 μM groups (91.7% vs. 87.8%, 88.8%; P<0.05) respectively. However, the blastocysts production was significantly higher in 100 μM group than others (26.2% vs. 13.6%, 14.0%, 18.2%; P<0.05) respectively. The optimal concentrations of flavone significantly enhanced the percentages of ICM:TE than control group (43.8% vs. 37.6%; P<0.05) accompanied with significantly higher expression levels of reprogramming related genes. In conclusion, the optimal concentrations of 1 μM during IVM and 100 μM during IVC can significantly improve the production of porcine in-vitro embryos.

Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).

To obtain in vivo matured oocytes for dog cloning, serum progesterone (P4) level were employed for ovulate determination. Radioactive immunoassay (RIA) is a traditional serum hormone assay method with highly radioactivity. The aim of this study was to evaluate the reliability of RIA and to compare its canine serum P4 concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). To obtain in vivo matured oocytes for canine somatic cell nuclear transfer, serum P4 levels were accurately measured with both methods of RIA and ECLI. Although both methods detected similar P4 level before ovulation, the mean P4 concentration using ECLI was significantly higher than that using RIA from 3days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of P4 were criteria for determination of ovulation. On other hand, high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was criteria for ovulation determination. To determine whether in vivo oocytes obtained by ECLI method could be used for canine cloning, six canines were selected as oocyte donors and two puppies were produced after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.