• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.46-46
• /
• 2003

Interleukin-10 (IL-10) is a homodimeric protein with a wide spectrum of anti-inflammatory and immune activities. It inhibits cytokine production and expression of immune surface molecules in various cell types. The transgenic mice carrying the human IL-10 gene in conjunction with the bovine $\beta$-casein promoter produced the human IL-10 in milk during lactation. Transgenic mice were generated using a standard method as described previously. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues with a primer set. In this study, stability of germ line transmission and expression of IL-10 gene integrated into host chromosome were monitored up to generation F15 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to F15 mice showed similar transmission rates (66.0$\pm$20.13%, 61.5$\pm$16.66%, 41.1$\pm$8.40%, 40.7$\pm$20.34%, 61.3$\pm$10.75%, 49.2$\pm$18.82%, and 43.8$\pm$25.91%, respectively), implying that the IL-10 gene can be transmitted stably up to long term generation in the transgenic mice. For ELISA analysis, IL-10 expression levels were determined with an hIL-10 ELISA and a mIL-10 ELISA kit in accordance with the supplier's protocol. Expression levels of human IL-10 from milk of generation F9 to F13 mice were 3.6$\pm$1.20 mg/ml, 4.2$\pm$0.93 mg/ml, 5.7$\pm$1.46 mg/ml, 6.3$\pm$3.46 mg/ml, and 6.8$\pm$4.52 mg/ml, respectively. These expression levels are higher than in generation F1 (1.6 mg/ml) mice. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations, although there was a little fluctuation in the transmission frequency and expression level between the generations.

• Korean Journal of Animal Reproduction
• /
• v.25 no.4
• /
• pp.305-315
• /
• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.

• Journal of Embryo Transfer
• /
• v.30 no.3
• /
• pp.207-211
• /
• 2015

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ${\beta}-actin$ promoter-driven firefly improved luciferase cDNA (${\beta}-act/luc^+$) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (${\beta}-act/luc^+$) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.5
• /
• pp.745-753
• /
• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.377-384
• /
• 2002

To investigate the expression patterns of proteins and growth factor signals in differentiated rabbit embryonic stem (ES) cells, ES cells with confluent stage grown of feeder layer and differentiated cells into embryoid bodies (EB) without feeder cell were applied to protein gel and Western blotting analysis. There were 66kDa and 28kDa specifically expressed in differentiated ES cell but not in undifferentiated ES cell while 25kDa protein band showed up in only undifferentiated ES cells. Also there were some difference of protein bands in several area of gel between differentiated and undifferentiated ES cells such as about 100 kDa, 50kDa and 27kDa areas, but there was no difference in band pattern of one-dimensional gel analysis between mouse ES cells and rabbit ES cells. IGF-I receptor and EGF receptor were expressed in differentiated cells and undifferentiated cells. And ICF-I and EGF were not expressed in both differentiated and undifferentiated cells. These results indicated that ES cells express their own proteins to inhibit differentiation while EB cells synthesize different proteins to differentiate, and 16F-I receptor and EGF receptor were expressed in both ES and EB cells probably for the different functions.

• Annals of Clinical Neurophysiology
• /
• v.7 no.2
• /
• pp.101-106
• /
• 2005

Background: There is no currently effective treatment for amyotrophic lateral sclerosis (ALS), although this disorder is a progressive neurodegenerative disease resulting in death within several years. Because recent evidence suggests that homocysteine (HC) is highly related to neurodegenerative disorders with aging, we tried to elucidate the effects of multi-vitamin therapy on G93A SOD1 transgenic mice. Methods: We treated this murine model of ALS with multi-vitamin (folic acid 1.97 mg/day, pyridoxine 0.98 mg/day, cyanocobalamin 0.1 mg/day) from 45 days of age, per oral. We performed the rotarod test from postnatal $10^{th}$ week, weekly. Results: We found that multi-vitamin reinforcement significantly prolonged average lifespan and delayed disease onset with improvement of motor performance. However, it did not significantly slow disease progression and statistical differences of weight loss were not observed between in transgenic mice and controls. Conclusions: These results suggest that multi-vitamin can be a potent therapeutic strategy for familial forms of ALS.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.215-216
• /
• 2001

Mouse monoclonal antibody(mAb) with an antigen specificity for major histocompatibility complex class Il(MHC class II) was produced and secreted from tobacco cell suspension culture by successive sexual crossesu. Expression and secretion of assembled antibody was observed in transgenic tobacco cell suspension culture by wetern blot analysis.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.58-58
• /
• 2002

Currently, the technique of pronuclear microinjection is the most successful and most widely-used method for producing transgenic animals. Among this technique, surperovulation and embryo transfer are the crucial steps for obtaining a large number of fertilized eggs and birth as much as potential founders from the transferred embryos. (omitted)

• Toxicological Research
• /
• v.24 no.3
• /
• pp.175-182
• /
• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.60-60
• /
• 2002

본 연구는 사람의 조혈촉진유전자가 형질전환된 돼지의 유즙내 hEPO 발현농도를 확인하고 3종의 동물 (mouse, rat, swine)에서 hEPO 의 생리활성을 분석하기 위하여 실시하였다. 새롬이 Fl 암컷의 유즙을 원심분리하여 지방을 제거한 후 hEPO 의 농도를 측정하고, PBS 로 유즙내 hEPO 농도가 100 IU/㎖가 되도록 희석하였다. Mouse (ICR)는 피하주사로 20 IU씩 2일 간격으로 3회 (0, 2, 4 일차 ) 주사하였으며, 주사 후 4회에 걸쳐 (0, 2, 4, 7 일차) 혈액을 채취하여. 분석하였다. (중략)