In all mammalian species, progesterone is essential in the preparation for and maintenance of pregnancy, if it occurs. Progesterone primes the endometrium for possible implantation and inhibits uterine contraction until birth. 20-alpha hydroxysteroid dehydrogenase (20α-HSD; EC.1.1.1.149) enzyme belongs to the family of aldo-keto reductases. 20α-HSD predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone (20α-OHP), and plays a crucial role in the termination of pregnancy and initiation of parturition. In addition, the activity of 20α-HSD during the luteal phase known to be inhibited by prolactin. In this study, we focused on the analysis of transgenic mice expressing EGFP under control of monkey 20α-HSD promotor in mice testis. The protein expression and localization were detected by Western blotting and Immunohistochemical analysis, respectively. 20α-HSD protein was detected at molecular weight of 37-kDa by Western blotting analysis and EGFP was found at 27-kDa in the testis of TG mice. Also EGFP and 20a-HSD protein expression on 1, 2, 4, 6 and 8 weeks after birth were assessed. Both of them were increased the expression level time-dependently. 20α-HSD were strongly expressed in seminiferous tubule from 1 week after birth as seen in Immunohistochemical analysis. However, EGFP was strongly expressed in the seminiferous epithelial cells. Then, we determined the expression of EGFP mRNA in mice testis. Using primers specific for mouse EGFP, mRNA expression levels were analyzed by RT-PCR. The EGFP molecular weights is 400bp, qRT-PCR results using EGFP primer, The Cq value of the ratio decreased as the age increased. On this basis, mRNA were increased the expression level time-dependently. In conclusion, these observations suggest that the 20α-HSD in testis could be play a pivotal role in the spermatogenesis.

Enhanced green fluoresce protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method in this study. The ERL836 transformants were generated with pCambia-egfp binary vector. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% of the fungal putative transformants were inserted by the T-DNA fragment. Of these transformants, 33.33% (2 transformants) expressed the egfp gene. The egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a reference for foreign protein expression in B. bassiana by using the AtMT method.

<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.

본 연구는 돼지 B-casein 유전자 위치에서 EGFP가 발현될 수 있는 knock-in 벡터를 구축하기 위하여 실시되었다. 돼지의 B-casein 유전자를 이용하여 knock-in 벡터를 구축하기 위해 돼지의 태아 섬유아세포로부터 B-casein 유전자를 동정하였고 EGFP, SV4O polyA signal을 동정하였다. Knock-in 벡터는 5' 상동 영역 약 5 kb와 3' 상동 영역 약 2.7 kb로 구성되어있으며, positive selection marker로 neor 유전자를, negative selection marker로 DT-A 유전자를 사용하였다. 구축된 knock-in 벡터로부터 EGFP의 발현을 확인하기 위하여 생쥐 유선 세포인 HC11 세포에 knock-in 벡터를 도입하였다. 그 결과 EGFP의 발현을 HC11 세포에서 확인하였다. 이와 같은 결과로서 이 block-in 벡터는 knock-in 형질전환 돼지를 생산하는데 사용될 수 있을 것으로 생각된다.

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

배아줄기세포는 외부 기인 특정 요소를 이용한 세포 조절물질 활성을 통하여 원하는 세포형태로 분화될 수 있다. 배아줄기세포의 이용성세포침투성단백질(CPP)은 몇 개의 아미노산으로 구성된 작은 펩타이드로 유용한 물질 수송계 중의 하나로 인식되고 있다. 본 연구에서는 몇 종류의 CPP를 이용하여 특정 단백질을 생쥐배아줄기세포(R1)내로 트렌스펙션하는데 있어 그 정도를 결정할 수 있는 요소들의 영향을 분석하였다. CPP인 Buforin II, pEP-1을 강도