The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

Freezing of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification of bovine expanded, hatched and SCNT embryos on the survival rate, apoptosis index and further development after thawing. Expanded, hatched and SCNT embryos were vitrified after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. Artificial shrinkage of the blastocyst was achieved after pushing a pulled pasteur pipet into the blastocoele cavity until it contracted. The shrunken and not shrunken embryos were exposed to cryoprotectant solution in 7.5% ethylene glycol-7.5% DMSOPBS with 20% FBS for 5 min. They were placed in a small volume of vitrification solution (15% ethylene glycol+15% DMSO+PBS+20% FBS+0.5 M sucrose) and plunged into liquid nitrogen on a cryotop. Then, after thawing, cryoprotectant was diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min. Under the optimal conditions, overall efficiency of the survival rate of bovine expanded, hatched, SCNT embryos in artificial shrinkage groups was higher compared with non-artificial shrinkage groups (p< 0.05). Especially, the numbers of TUNEL-positive nuclei in artificial shrinkage groups were significantly reduced than those of non-artificial shrinkage groups among frozen-thawed expanded, hatched, and SCNT blastocysts (p< 0.05). Our results showed that survival rates in cryopreserved expanded, hatched, SCNT embryos could be improved by reducing the fluid content. Therefore, we suggest that artificial shrinkage method is a effective pretreatment technique for the cryotop vitrification of expanded, hatched, SCNT bovine blastocysts.

<Objective> Injection of a linear transgene into male pronucleus has been widely used to produce Transgenic (Tg) mice. This approach however is inefficient and results in concatemerised transgene insertion and associated reduced protein expression from such insertions. The objective of this study is to develop active transgenesis method by using a piggyBac transposase plasmid DNA, and generate double transgene harboring transgenic mice. <Method> We examined the piggyBac transposase plasmid (pm- GENIE‐3) on its ability to produce transgenic animals with NaOH, HCl and FuGENE6 treated sperm followed by ICSI‐Tr, for its effectiveness in creating EGFP Tg mice, as judged by offspring epifluorescence. After these steps, we explored if the embryo development affects ICSI‐Tr efficiency by using substrate‐free media or aphidicolin. Moreover, we tested to determine if transgenesis is possible by directly injecting the DNA into the cytoplasm or into pronuclei. Finally, we attempted the introduction of two transgenes, such as EGFP and dsRED simultaneously in one transposon and the ability to generate double Tg mice by using NaOH treated sperm during ICSI‐Tr. <Results> The best results were obtained when sperm were treated with NaOH and co‐incubated with circular plasmid DNA of pmGENIE‐3. This resulted in Tg pups that could successfully express EGFP, with efficiencies of 37.9% of born animals being transgenic. Furthermore, the effectiveness of this method was proved by the production of Tg offspring from inbred strains of mice, such as C57BL/6, Balb/c and CD‐1 nude. While injection of DNA into the pronucleus or cytoplasm of one cell embryos, and delayed embryo development‐method were not as effective as ICSI‐Tr in producing Tg mice, they nevertheless proved successful. Finally, NaOH‐ICSI‐Tr successfully obtained Tg mice expressing both the EGFP and dsRED transgene. In conclusion, the current study developed an active form of NaOH‐ICSI‐Tr mediated transgenesis utilizing the piggyBac transposition machinery, and was successful in obtaining Tg mice which expressed simultaneously not only EGFP but also the dsRED transgene stably inserted in these animals.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.

The green peach aphid (Myzus persicae) is a serious pest of agricultural and horticultural crops all over the world. M. persica has rapidly developed resistance to a wide variety of insecticides, including carbamates. The E4/FE4 carboxylesterase is known to be involved in carbamate resistance. To compare the E4/FE4 carboxylesterase gene copy number, as a genetic resistance marker, between seven field strains, quantitative real-time PCR (qPCR) was performed. In addition, quantitative sequencing (QS) was employed to predict the frequencies of acetylcholinesterase (AChE) mutations (A301S and S431F) that are associated with target site insensitivity. All M. persica strains examined possessed the S431F mutation in the heterozygous state except for a susceptible strain, implying the possibility of AChE duplication. In contrast, no A301S mutation was found. Frequency prediction equation was generated from the plots of signal ratios and amplification critical time, which showed a high correlation (r2>0.996). QS analysis of M. persicae populations revealed that the allele frequency of S431F ranged 4% to 63%. Taken together, the AChE resistance allele frequencies determined by QS and the E4/FE4 gene copy number by qPCR should facilitate the detection and monitoring of carbamate resistance in M. persicae in the field.

The screening of effective lethal genes was conducted via the systemic delivery of dsRNA for the RNAi-based management of Tetranychus urticae. Six candidate genes (COPI coatmer, T_COPI; ESCRT III_Snf7, T_SNF7; Ribosomal protein S4, T_RPS4; v-ATPase A subunit 2, T_V-ATPase; Aminopeptidase N, T_APN3; Acetylcholinesterase, T_AChE) and two reference genes (EGFP and T_AChEintron) were tested for the experiment. The permeated dsRNA to the leaf disc (ca. 30 mm diameter) was detected at 6 h after treatment, indicating that dsRNA could move through veins on the leaf. In the reference gene selection, the T_AChEintron was chosen for its low mortality compared with EGFP gene. In the evaluation of mortality, the T_COPI, T_V-ATPase and T_RPS4 exerted higher toxicities at 24 hour after treatment among six genes tested. Interestingly, T_APN3 showed toxicity after 72 hour. In summary, the dsRNA delivery via leaf disc was effective in screening lethal genes and some genes, such as COPI, V-ATPase and RPS4, can be applicable for establishing a RNAi-based control system against T. urticae.