• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.239-246
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• 1992

Capacitated and acrosome~reacted spermatozoa were microinjected into the perivitelline space of bovine oocytes matured in vitro. Oocytes obtained from the ovaries of slaughtered heifers and cows were cultured in vitro in the TCM-199 supplemented with 20% FCS for 24 hr at 39$^{\circ}C$ under an atmosphere of 5% CO$_2$ 8% O$_2$. Fresh or frozen spermatozoa were incubated for 2 hr at 39°C under an atmos-phere of 5% CO$_2$, 8% O$_2$ in Ham's F-lO medium containing 0.75% BSA for capacitation, and kept for 30 min in culture medium containing 12 mM of dbcGMP and lOmM of immidazol for acrosome resction. One motile spermatozoon was injected into the perivitelline space of each oocyte. The 2nd polar body and the pronuclei were observed in 9.5% and 5.4% of oocytes, respectively. The rate of cleavage of oocyte over 2-cell stage was 4.1%(10 of 242), These results indicate that the microinjection may be a useful technique to study sperm-oocyte interaction.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.603-611
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• 1997

The motor evoked potentials (MEPs) have been advocated as a method of monitoring the integrity of spinal efferent pathways in various injury models of the central nervous system. However, there were many disputes about origin sites of MEPs generated by transcranial electrical stimulation. The purpose of present study was to investigate the effect of major extrapyramidal motor nuclei such as lateral vestibular nucleus (VN) and medullary reticular nucleus (mRTN) on any components of the MEPs in adult Sprague-Dalwey rats. MEPs were evoked by electrical stimulation of the right sensorimotor cortex through a stainless steel screw with 0.5mm in diameter, and recorded epidurally at T9 - T10 spinal cord levels by using a pair of teflon-coated stainless steel wire electrodes with 1mm exposed tip. In order to inject lidocaine and make a lesion, insulated long dental needle with noninsulated tips were placed stareotoxically in VN and mRTN. Lidocaine of $2{\sim}3\;{\mu}l$ was injected into either VN or mRTN. The normal MEPs were composed of typical four reproducible waves; P1, P2, P3, P4. The first wave (P1) was shown at a mean latency of 1.2 ms, corresponding to a conduction velocity of 67.5 m/sec. The latencies of MEPs were shortened and the amplitudes were increased as stimulus intensity was increased. The amplitudes of P1 and P2 were more decreased among 4 waves of MEPs after lidocaine microinjection into mRTN. Especially, the amplitude of P1 was decreased by 50% after lidocaine microinjection into bilateral mRTN. On the other hand, lidocaine microinjection into VN reduced the amplitudes of P3 and P4 than other MEP waves. However, the latencies of MEPs were not changed by lidocaine microinjection into either VN or mRTN. These results suggest that the vestibular and reticular nuclei contribute to partially different role in generation of MEPs elicited by transcranial electrical stimulation.

• The Korean Journal of Pharmacology
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• v.23 no.2
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• pp.77-85
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• 1987

This study was carried out to determine the role of cholinoceptors in the ventrolateral medulla on central control of blood pressure (BP) and heart rate (HR). In rats anesthetized with urethane and paralyzed, microinjections of the neuroexcitatory amino acid L-glutamate (300 ng/site) were performed to functionally identity the vasopressor area (VLPA) and the vasodepressor area (VLDA) in the ventrolateral medulla oblongata. 1. The bilateral microinjection of carbachol (300 ng/site) into the VLPA produced significantly an increase in BP and HR which was not blocked by bilateral pretreatment of hexamethoium ($4\;{\mu}g/site$). 2. The bilateral microinjection of physostigmine (200 ng/site) and oxotremorine (300 ng/site) into the VLPA produced significantly an increase in BP respectively. 3. The bilateral microinjection of atropine ($4\;{\mu}g/site$) into the VLPA produced significantly a decrease in BP and HR. 4. The bilateral micro injection of acetylcholine (500 ng/site) and dimethylphenylpiperazinium (500 ng/site) into the VLDA produced significantly a decrease in BP and HR respectively. 5. The depressor and bradycardiac responses elicited by the bilateral microinjection of acetylcholine (500 ng/site) into the VLDA were blocked by bilateral pretreatment of hexamethonium ($4\;{\mu}g/site$). The results suggest that the activation of cholinoceptors in VLPA produce hypertensive and tachycardiac responses which may be mediated by muscarinic receptors, and the activation of cholinoceptors in VLDA produce hypotensive and bradycardiac responses which may be mediated by nicotinic receptors.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.399-407
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• 2001

The microinjection of retroviral vectors into the perivitelline spaces of MII-stage oocytes increased production of transgenic bovine embryos. However, oocytes have various sizes of perivitelline space, and there is the tendency that the oocyte membranes are damageable by micropipettes during the injection of retroviral vectors into perivitelline spaces or oocytes. Thus, it was not always possible to stably inject retroviral vector into perivitelline spaces of oocytes. Here we used sucrose to minimize the damage of the oocyte membrane. When the oocytes were suspended in 0.5% sucrose, poor quality oocytes showed rough cytoplasmic membranes, while good quality oocytes maintained smooth membranes. However, when the tatters were subjected to in vitro fertilization, no significant differences were observed in cleavage rates (82% of control Vs. 84% of sucrose treated oocytes). The Same trends were obtained from the oocytes fertilized after microinjection of LN$\beta$-EGFP and LNC-hGH genes into the perivitelline spares. The rates of cleavage and blastocyst from microinjection of LN$\beta$-EGFP genes were 81 and 25%, and from microinjection of LNC-hGM genes were 53 and 30%, respectively. The result indicated that microinjected oocytes could develop to the blastocyst stages after in vitro fertilization with no significant difference from control group. Moreover, the integration of hGH-gene (by PCR analysis) was detected in 52% of infected cleaved embryos and the expression of EGFP-gene (under a fluoresrence microscope) was also observed in 34% of infected blastocyst. These results indicated that 0.5% sucrose treatment could be an efficient method not only to select good quality embryos but also to inject retroviral vectors into perivitelline spares without any harm and hence improving developmental rates.

• The Korean Journal of Zoology
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• v.38 no.3
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• pp.340-347
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• 1995

We have used two kinds of P element constructs, Pc[(ry+)B] and p[(ry+)$\Delta$SX9], for genetic transformation by microinjection of D. melanogaster. Pc[(ry+)B] construct carrying the rosy gene within an autonomous P element was injected into a true M strain caring the ry506. mutation. The source of transposase for microinjection and transformation was provided by a P element helper plasmid designated p-$\Delta$2-3hs$\pi$, which was co-injected with nonautonomous P[(ry+)$\Delta$SX9] construct into same ry506 M strains. A dechorination method was adopted and 35 independent transformed lines were obtained froin 1143 G0 Injected (35/1143). About 20% of the injected embryos eclosed as adults. Among G0 eclosed flies, approximately 40% exhibited eye color that was similar to wild-type (ry+), but about 60% of fertile G0 transformed lines appeared to have no G1 transformants. Therefore it is unlikely that G0 expression requires integration of the rosy transposon into chromosomes. Pc[(ry+)B] and P[(ry+)$\Delta$SX9] constructs were found to be nearly same in the frequency of element-mediated transformation. On the basis of these results, nonautonomous P elements constructs could he used as same effective vectors in P element-mediated transformation for introducing and fixing genes in insect populations.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.95-104
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• 2002

This study was performed during the four seasons for the production of transgenic pigs containing the human erythropoietin(hEPO) transgene. Purebred Landrace gilts and sows approximately 8∼15 months of age (n=42) were used fur the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Synchronization and superovulation procedures were used that cyclic gilts were synchronized with 20mg altrenogest (ALT) per day for 9days after PG600 administration followed by superovulation with 1500IU pregnant mares serum gonadotropin (PMSG) and 500IU human chorionic gonadotrophin (hCG). Preparation of recombinant gene for microinjection is mice whey acidic protein promoter (mWAP) linked to human erythropoietin (hEPO) gene. After hormone treatment, 650 embryos were collected from 23 donors and 83.1% (540/650) embryos were in 1-cell stage which can be visualized the pronuclei for DNA microinjection. A total of 543 DNA microinjected embryos fiom donors were transferred to 19 synchronized recipients, seven of them maintained pregnancy and delivered 47 piglets. One of the 47 offsprings were determined to have transgene by PCR analysis. The overall rate of transgenic production was 2.13% (tansgenic/offspring). This study provides the success and useful information regarding production of transgenic pig for bioreactor research.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.87-94
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• 2002

This study was performed during the four seasons for the production of transgenic pigs containing the Cellulase Digest Gene. Purebred Landrace gilts and sows approximately 8∼15 months of age (n=126) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality fur zygote collection. Synchronization and superovulation procedures were used that cyclic gilts were synchronized with 20mg altrenogest (ALT) per day for 9 days after PG600 administration followed by superovulation with 1000 IU pregnant mares serum gonadotropin (PMSG) and 750IU human chorionic gonadotrophin (hCG). The cellulase digestion gene for microinjection is rat elasterase promoter (rEl) linked to CelD gene. After hormone treatment, 1,422 embryos were collected from 91 donors and 95.6% (1,359/1,422) embryos were in 1-cell stage which can be visualized the pronuclei for DNA microinjection. A total of 725 DNA microinjected embryos transferred into 35 recipients and produced 65 piglets from 13 litters. Pregnancy rate according to the number of transferred embryos to recipients was higher the group which received 21 to 24 embryos (50.0%) than other groups 20.0% in less and 33.3% in more. A tail tissue was collected from 65 piglets for biopsy. PCR screening was performed on each DNA sample using two separate sets of primers specific for the 5'- and 3'-flanking region of the rEl-CelD gene. Five of the 65 piglets (7.69%) were positive for the transgene. This study provide useful information regarding production of transgenic pig for bioreactor research.

• Animal Bioscience
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• v.36 no.8
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• pp.1180-1189
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• 2023

Objective: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. Methods: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. Results: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. Conclusion: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

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

• Proceedings of the Korean Society of Precision Engineering Conference
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• 2010.05a
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• pp.963-964
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• 2010