This study was to analyse the usability of morphological evaluation of vitrified-thawed oocyte before somatic cell nuclear transfer (SCNT) using Oosight imaging system to show spindle. For the vitrification, in vitro matured bovine MII oocytes were treated by two-step freezing medium without (control group) or with 5 ug/ml cytochalasin-b (CCB group). In Exp. 1, after thawing, recovered oocytes in each treatment group were assessed by live image using Oosight imaging system or/and cytoskeletal protein image using immunostaining. In Exp. 2, in each treatment group the in vitro developmental potential of frozen-thawed bovine oocytes post evaluation using Oosight imaging system and then SCNT was investigated. The SCNT embryos were cultured in CR1aa medium supplemented with 10% FBS, 1 mM EGF and 1 mM IGF at 38.5 C in 5% O2 and 5% CO2 in air for 8 days. In Exp.1, the rates of in vitro survival, morphological good grade and spindle normality of CCB treatment group (91.1%, 54.2% and 55.5%) were better than those of control group (86.1%, 48.5% and 48.5%). After SCNT using vitrified-thawed oocyte, the rates of fusion, reconstructed embryos and blastocyst development were also high in CCB treatment group (66.6%, 36.4% and 3.0%) than control group (60.0%, 27.3% and 0%). These results demonstrated that the identification of morphological spindle image of the vitrified-thawed bovine oocytes using Oosight imaging system helps to predict the SCNT embryo quality.

The production of transgenic animals using somatic cell nuclear transfer (SCNT) has been widely described. A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene which occasionally results in serious physiological disorders in the transgenic animal. In this study, we designed three different expression vectors that express the hEPO gene. hEPO is a hormone produced by the kidney that promotes the formation of red blood cells by the bone marrow. For the in vitro production of transgenic embryos, the different expression vectors were transduced into holstein ear fibroblast cells, respectively, and GFP expressed donor cells were transferred into enucleated oocytes, and then the reconstructed SCNT embryos were developed into pre-implantation stage. From three replicates, GFP expressed 112 transgenic SCNT embryos were produced. When their cleavage rate and blastocyst rate were compared with non-transgenic SCNT embryos, the results were presented into 73.2% vs. 76.9% and 26.8% vs. 30.6%, respectively, there were no differences. Also, total cell number and ICM cell numbers of day 8 blastocysts were statistically not different between the transgenic SCNT groups (120.6±7.9 and 31.4±8.2) and control SCNT group (128.3±4.8 and 35.3±4.0). The GFP expression levels were presented consecutively high during the culture of transgenic SCNT embryos. By analysis of semi-quantitative RT-PCR, the relative expression levels of hEPO mRNA and pluripotent gene were determined. These results demonstrated that the hEPO expressed transgenic bovine embryos can be efficiently produced in vitro by SCNT technique, while their potential of cloned animal production have to be examined in further study.

Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem (ES) cells. Differentiation capacity of the parthenogentic ES cells was rather lower than that of fertilized embryos derived ES cells, which might be the result of the absence of male genome. However, parthenogenetic ES cells might be useful research tool for genetic engineering and generating SCNT embryo derived ES cells. In our previous study, we reported that establishment of several bovine ES cell lines from in vitro fertilized (IVF) embryos named JNU-ibES. Based on this data, the objective of this study is to generate parthenogenetic ES cells and to examine their stem cell characteristics. Total 107 parthenogenetic embryos produced at day 8 or 9 were classified into their developmental stages (full expanded x 40, hatched x 67). For producing ES cells, ICM and trophetoderm-rich clumps were mechanically dissociated and were cultured on mitomycin- C treated mouse embryonic fibroblast feeder cell drop and covered with mineral oil in DMEM medium containing 20% FBS, 5 ng/ml basic FGF, 1% nonessential amino acids, and 0.55 mM b-mercaptoethanol. We obtained 20 primary parthenogenetic bovine ES (pbES)-like cell colonies. And pbES colony formation was higher in hatched blastocyst (25.4%, 16/67) than expanded blastocysts (10%, 4/40). Among those colonies, 5 pbES cell lines were successfully established and they were named as a series of JNU-pbES. These pbES cells were positively expresssed pluripotency markers such as Oct4, Nanog, TRA-1-81, SSEA-1 and alkaline phosphatase. This result demonstrated that the establishment efficiency and characteristics of pbES cell line was very similar to those of ibES cell line.

The leaf beetle, Chrysolina aurichalcea (Coleoptera: Chysomelidae), is a pest damaging plants of Compositae. In order to understand the genetic diversity and geographic variation we sequenced a portion of mitochondrial COI gene (658 bp) and complete nuclear internal transcribed spacer 2 (ITS2) of the species collected from seven Korean localities. A total of 17 haplotypes (CACOI01 ~ CACOI17), with the maximum sequence divergence of 3.04% (20 bp) were obtained from COI gene sequence, whereas 16 sequence types (ITS2CA01 ~ ITS2CA16), with the maximum sequence divergence of 2.013% (9 bp) were obtained from ITS2, indicating substantially larger sequence divergence in COI gene sequence. Phylogenetically, the COI gene provided two haplotype groups with a high nodal support (≥ 87%), whereas ITS2 provided one sequence type group with a high nodal support (≥ 92%). The result of COI gene may suggest the presence of historical biogeographic barriers that bolster genetic subdivision in the species. Different grouping pattern between COI gene and ITS2 sequences were interpreted in terms of recent dispersal, reflected in the ITS2 sequence. Finally, finding of unique haplotypes and sequence types only from Beakryeng-Islet population was interpreted as an intact remnant of ancient polymorphism. As more samples are analyzed using further hyper-variable marker, further fruitful inference on the geographic contour of the species might be available.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.

The phylogenetic relationships among the Nymphalidae (Lepidoptera: Papilionoidea) have been controversial in several perspective. The present study sequenced a total of ~ 3,500 bp from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) in 80 nymphalid species belonging to seven subfamilies (Linmenitidinae, Heliconiinae, Nymphalinae, Apaturinae, Libytheinae, Satyrinae, and Danainae), along with those of six lycaenid species as outgroups. Phylogenetic analyses via Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms concordantly supported the subfamilial relationships of (((((Linmenitidinae + Heliconiinae) + (Nymphalinae + Apaturinae)) + Libytheinae) + Satyrinae) + Danainae), with high nodal support for monophyletic subfamilies and tribes. This result is largely consistent with a previous study performed with a substantially large sequence information and morphological characters, except for the position of Libytheinae that has previously been placed as the sister to all reminder of Nymphalidae.

The complete mitogenome (20,456 bp) of Challia fletcheri (Dermaptera: Pygidicranidae) as the first dermapteran insect is the longest among sequenced insects. The genome contained typical gene sets, but harbored the largest TRU among Exopterygota and Palaeoptera. The AT- and GC-skewness showed more Ts and Gs encoded on the major strand, whereas more As and Cs on the minor strand, presenting a reversal to the general pattern found in most insect mitogenomes. This pattern was explained in terms of inversion of replication origin. The gene arrangement of C. fletcheri genome is unique in insects and differs from the ancestral type found in insects by a series of gene translocations and/or inversions. We hypothesize that the markedly different gene arrangement is probably due to some unique organism-level properties, which allow relaxed selection against mitochondrial gene rearrangement. All phylogenetic analyses consistently placed Orthoptera as the sister to the group composed of a monophyletic Isoptera + Mantodea + Blattodea and a monophyletic Grylloblattodea + Mantophasmatodea + Phasmatodea, and placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera.

The Samia cynthia ricini (Lepidoptera: Saturniidae) is a commercial silk-producing insect belonging to an insect family Saturniidae in Bombycoidea. The species that has presumably been originated in India, is distributed in India, China, and Japan. Unlikely domestic silkworm the prime host plant for the species is a castor-oil plant (Ricinus communis in Euphorbiaceae). Recently, the eri-silkworm also is reared in Korea and is expected to be utilized for a diverse purpose. In this report, we present the complete mitochondrial genome of the species with the emphasis of a few major characteristics. The 15,384-bp long S. cynthia ricini (Lepidoptera: Saturniidae) mitochondrial genome was amplified into three long overlapping fragments (from COI ~ ND4, ND5 ~ lrRNA, and lrRNA ~ COI) and subsequent several short fragments using the long fragments as temperate. The primers for both long and short fragments were designed solely for lepidopteran genomes, without any species-specific primers. As a usual the genome is composed of 37 genes: 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, and one large non-coding region termed the A+T-rich region. Arrangement of the genome is identical to those of other lepidopteran mitochondrial genome, but this differs from the common arrangement found in a diverse insect order, by the movement of tRNAMet to a position 5’- up stream of tRNAIle. Unlikely previous report on the start codon for COI gene in Lepidoptera S. cynthia ricini COI gene starts with typical ATT codon located between tRNATyr and the beginning region of COI gene. The 22 tRNAs that are interspersed throughout the mitogenome ranged in length from 62 to 71 bp. All tRNAs but tRNASer(AGN) were shown to be folded into the expected cloverleaf secondary structures. More detailed structural and phylogenetic analyses among Bombycidae and Saturniidae in connection with other families in the Bombycoidea will be performed soon

The complete mitochondrial genome sequence of the nerippe fritillary butterfly, Argynnis nerippe, which is listed as an endangered species in Korea, is described with an emphasis on the A+T-rich region. The 15,140-bp long circular molecule consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one control region, known in insect as the A+T-rich region, as found in typical metazoans. The 329-bp long A+T-rich region located between srRNA and tRNAMet possessed the highest A/T content (95.7%) than any other region of the genome. Along with the several conserved sequences found typically in the lepidopteran insects the genome contained one tRNAMet-like and tRNALeu(UUR) -like sequence in the A+T-rich region.