• The Korean Journal of Pain
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• v.35 no.3
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• pp.261-270
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• 2022

Background: The rostral ventromedial medulla (RVM) is a critical region for the management of nociception. The RVM is also involved in learning and memory processes due to its relationship with the hippocampus. The purpose of the present study was to investigate the molecular mechanisms behind orexin-A signaling in the RVM and hippocampus's effects on capsaicin-induced pulpal nociception and cognitive impairments in rats. Methods: Capsaicin (100 g) was applied intradentally to male Wistar rats to induce inflammatory pulpal nociception. Orexin-A and an orexin-1 receptor antagonist (SB-334867) were then microinjected into the RVM. Immunoblotting and immunofluorescence staining were used to check the levels of cyclooxygenase-2 (COX-2) and brain-derived neurotrophic factor (BDNF) in the RVM and hippocampus. Results: Interdental capsaicin treatment resulted in nociceptive responses as well as a reduction in spatial learning and memory. Additionally, it resulted in decreased BDNF and increased COX-2 expression levels. Orexin-A administration (50 pmol/1 µL/rat) could reverse such molecular changes. SB-334867 microinjection (80 nM/1 µL/rat) suppressed orexin's effects. Conclusions: Orexin-A signaling in the RVM and hippocampus modulates capsaicin-induced pulpal nociception in male rats by increasing BDNF expression and decreasing COX-2 expression.

• Korean Chemical Engineering Research
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• v.47 no.5
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• pp.538-545
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• 2009

The principle and application of a scanning probe microscopy(SPM) are reviewed briefly, and a low-invasive single cell manipulation and a gene delivery technique using an etched atomic force microscopy(AFM) probe tip, which we call a nanoneedle, are explained in detail. The nanoneedle insertion into a cell can be judged by a sudden drop of force in a force-distance curve. The probabilities of nanoneedle insertion into cells were 80~90%, which were higher than those of typical microinjection capillaries. When the diameter of the nanoneedle was smaller than 400 nm, the nanoneedle insertion into a cell over 1 hour had almost no influence on the cell viability. A highly efficient gene delivery and a high ratio of expressed gene per delivered DNA compared the conventional major nonviral gene delivery methods could be achieved using the gene modified nanoneedle.

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

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.856-860
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• 2000

Cloning technology continues to capture widespread attention by the international news media and biomedical and agricultural industries. The future uses of this technology could potentially contribute to major advances in biomedical and agricultural sciences. Cloned transgenic dairy cattle possessing milk promoters directing transgenes will produce pharmaceutical proteins in their milk faster, more efficiently and less expensively than transgenic cattle created using microinjection techniques. Additionally, cloned transgenic fetuses and animals may become a source of cells, tissue and organs for xenotransplantation. Lastly, but maybe most importantly, enhanced production traits and disease resistance may be realized in animal agriculture by utilizing these new technologies. The recent advances in the cattle cloning technology are important but there are still major obstacles preventing widespread commercial use of this technology. The type of donor nucleus, recipient cytoplasm, and cloning procedures used will impact the potential number of clones produced and the uses of the technology. In addition, the new advances in cloning methodology have not improved the relatively low pregnancy rates or reduced the incidence of health problems observed in cloned offspring. These problems may require novel techniques to decipher their cause and new methods of preventing and/or diagnosing them in the preimplantation embryo. The commercial potential is enormous for cloning technology; however, little has been done to improve the efficiencies of the procedure. Improving procedural efficiencies is a critical developmental milestone especially for potential uses of cloning technology in animal agriculture.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.35-41
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• 1995

In this study, we have tested whether the retrovirus vector system is applicable in transgenic cattle production. To overcome low infectivity of currently available retrovirus vector system we have directly microinjected retrovirus-producing cells into the perivitelline space of the day 1.5 embryos. The virus-producing cell line was designed to release replication-defective retrovirus encapsidated with Gibbon ape leukemia virus (GaLV) envelope protein. E. coli LacZ gene was used as a marker gene to facilitate evaluation of the transgene expression and X-gal staining at morula or blastocyst stage resulted in expression of E. coli LacZ gene The results in these experiments were summarized as follows : 1. The lowest concentration of polybrene necessary for efficient virus infection was Sf' g/ml. 2. Development rate from day 1.5 embryos microinjected with virus-producing cells to the morulae /blastocysts was 29%. 3. 21% of the morulae /blastocysts were LacZ+. 4. There was no evidence that the retrovirus-producing cells used in this study produced replication-competent retrovirus.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.89-89
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• 2003

본 연구에서는 1.7kb 및 3.1kb bovine $\beta$-casein promoter의 유전자 발현 조절능력을 알아보기 위해 1 7kb 및 3.1kb bovine $\beta$-casein promoter에 human Type II Collagen 유전자를 연결해서 DNA microinjection으로 형질전환생쥐를 생산하였다. 총 8마리의 founder생쥐(1.7kb collagen : 5마리, 3.1kb collagen 3마리)를 생산하였고 이 founder생쥐와 wild type 생쥐를 mating시켜서 $F_1 및 F_2$ 새끼를 얻었다. $F_1 및 F_2$새끼들에서 human Type II collagen 유전자의 transmission rate는 약 50%로 Mendel의 법칙에 따라 분리되어 안정적으로 유전자가 염색체에 정착되어 있음을 확인하였다. 이들 $F_1 및 F_2$새끼 중 암컷들을 임신시켜 분만 후 5-10 일경에 유선조직을 포함하여 여러 조직으로부터 RNA를 추출하여 Northern blotting 및 RT-PCR 방법을 이용하여 Type II collagen mRNA의 발현을 분석하였다. 유선에서의 발현은 1 7 kb 및 3.1 kb line별로 각각 1 line씩 발현되지 않았고, 그 외 line에서는 모두 발현되는 것으로 확인되었다. 유선에서의 Type II collagen mRNA 발형양은 1.7 kb 및 3.1 kb bovine $\beta$-casein promoter사이에서는 큰 차이를 나타내지 않았으나 1.7 kb promoter 형질전환생쥐의 경우 유선 이외 조직에서도 발현되는 양상을 나타내었고, 3.1kb promoter line에서는 유선특이적으로 발현시키는 양상을 나타내었다. 그러므로 bovine $\beta$-casein promoter의 1.7 kb와 3.1 kb 사이에 유선특이적 발현을 유도하는 조절부위가 있을 것으로 추정된다.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.4
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• pp.456-464
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• 2010

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.1-2
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• 2000

High production of milk and its components are necessary to allow maximal growth of developing piglets. In this study, transgenic pigs were produced containing the $\alpha$-lactalbumin gene, whose product is a potential limiting component in the production of milk. Two lines of transgenic pigs were produced to analyze the effects that overproduction of the milk protein $\alpha$-lactalbumin may have on milk production and piglet growth. Transgenic pigs were produced through microinjection of the bovine $\alpha$-lactalbumin gene. The gene construct contained 2.0 kb of 5 flanking region, the 2.0 kb coding region and 329 bp of 3 flanking region. Sows hemizygous for the transgene produced as much as 0.9 g of bovine $\alpha$-lactalbumin per liter of pig milk. The production of the bovine protein caused approximately a 50 % increase in the total $\alpha$-lactalbumin concentration in pig milk throughout lactation. The concentration of bovine $\alpha$-lactalbumin was highest on day 0 and 5 of lactation and decreased as lactation progressed. The ratio of bovine to porcine $\alpha$-lactalbumin changed during the sow's lactation. This ratio was 4.3 to 1 on day 0 of lactation, but by day 20 of lactation the ratio was 0.43 to 1. This suggested that the bovine transgene and the endogenous porcine gene were under slightly different control mechanisms. The higher level of total $\alpha$-lactalbumin present on day 0 of lactation was correlated with higher lactose percentage on day 0 in transgenic sows (3.8 %) as compared to controls (2.6 %) (P < 0.01). Although there was also a trend for higher lactose percentage in transgenic sows on day 5 and 10 of lactation, no significant differences were observed. These data suggest that $\alpha$-lactalbumin is limiting early in lactation of swine. Furthermore, higher concentrations of $\alpha$-lactalbumin early in lactation may boost milk output.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.85-90
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• 2001

To study the signaling effect of growth hormone (GH) in vivo on animal physiology, transgenic mice containing GH Receptor (GHR) gene fused to metallothionein promoter were produced by DNA microinjection into one-cell stage embryos. Three founder mice were produced with transgenic mice with approximately 4~6 copies of GHR genes and transgene was transmitted into the progeny. The founder mice were mated with normal mice to produce F$_1$ mice, and intergation and transmission of transgene were checked by polymerase chain reaction and Southern blot methods. Transmission rate of GHR transgenic mice were 20~50% in F$_1$ generation and 50% in F$_2$ generation which means that some founder mice were mosaic and transgene in F$_1$ mice was transmitted to F$_2$ progeny with Mendelian ratio. Death rate of GHR transgenic mice after birth was about 10~30% in F$_1$ and F$_2$ progenies indicating that GHR gene may affect death of transgnenic progeny.