• Title/Summary/Keyword: DNA separation

Search Result 121, Processing Time 0.028 seconds

Studies on the Efficient Separation of Primordial Germ Cells and Introduction of Foreign DNA in the Chicken (닭에서 원시생식세포의 효율적 분리 및 외래 유전자 전이에 관한연구)

  • 정동기;한재용
    • Proceedings of the Korea Society of Poultry Science Conference
    • /
    • 1999.11a
    • /
    • pp.11-33
    • /
    • 1999
  • This study was conducted to determine the embryonic stages for the isolation of the highest number of PGCs and to improve PGCs enrichment method. The primordial germ cells(PGCs) from different sources of chick embryos were isolated. The embryonic stage having the highest number of PGCs from each sources was selected ; 1-day-old embryos for germinal crescent (stage 6-8), 2.5-day-old embryos for blood (stage 17-18) and 5.5-day-old embryos for gonad (stage 27-28). The number of PGCs from one embryonic germinal crescent, blood and gonad was about 87$\pm$1.8, 103$\pm$4.0, and 932$\pm$10.9, respectively. The viability of PGCs after Ficoll from each sources was similar, showing approximately 70%. the PGCs enrichment method was improved using Ficoll density gradient centrifugation. After this step the purity of PGCs from germinal crescent, blood, and gonad was 45$\pm$9.10%, 85$\pm$1.18%, and 86$\pm$0.19%, respectively. Also, PGCs were picked up by mouth pipette to improve the purity. This improved method for the separation of PGCs from different sources will serve as a useful too to preserve the foundation stocks of poultry and to produce germline chimeras.

  • PDF

Maternal Origins of the Jeju Native Pig Inferred from PCR-RFLP Haplotypes and Molecular Phylogeny for Mitochondrial DNA CYTB Gene Sequences (미토콘드리아 DNA CYTB 유전자 서열에 대한 분자 계통과 PCR-RFLP 반수체형에 근거한 제주재래돼지의 모계 기원)

  • Han, Sang-Hyun;Ko, Moon-Suck;Jeong, Ha-Yeon;Lee, Sung-Soo;Oh, Hong-Shik;Cho, In-Cheol
    • Journal of Life Science
    • /
    • v.21 no.3
    • /
    • pp.341-348
    • /
    • 2011
  • In an effort to gain greater understanding of the maternal lineages of the Jeju native pig (JNP), we analyzed the mitochondrial DNA (mtDNA) CYTB gene and compared it with those of other pig breeds. PCR-RFLP analysis was conducted with six pig breeds including JNP, and then the RFLP patterns allowed for the separation of the pig breeds into two distinct haplotypes (mtCYTB1 and mtCYTB2). The JNP CYTB sequences were detected in both the European and Asian breed clusters on the phylogenetic tree. The J2 group was sorted with the indigenous cluster of Asian pig lineages and was related closely to Chinese native pig breeds, but a second group, J1, was sorted with the European pig lineages and appeared to be related to Spanish Iberian native pigs, rather than to Asian breeds. These results indicate that the JNP currently raised on Jeju Island have two major maternal origins estimated in Asian and European pigs. We concluded that the JNP that share a common lineage with indigenous Asian pigs were domesticated in the distant past, originating from pigs that were already being raised elsewhere at that time, and that the European pig breeds introduced in the twentieth century have also contributed to the formation of this pig population.

Mitotic Cohesin Subunit Mcd1 Regulates the Progression of Meiotic Recombination in Budding Yeast

  • Lee, Min-Su;Yoon, Sang-Wook;Kim, Keun Pil
    • Journal of Microbiology and Biotechnology
    • /
    • v.25 no.5
    • /
    • pp.598-605
    • /
    • 2015
  • The cohesin complex holds sister chromatids together and prevents premature chromosome segregation until the onset of anaphase. Mcd1 (also known as Scc1), the α-kleisin subunit of cohesin, is a key regulatory subunit of the mitotic cohesin complex and is required for maintaining sister chromatid cohesion, chromosome organization, and DNA repair. We investigated the function of Mcd1 in meiosis by ectopically expressing Mcd1 during early meiotic prophase I in Saccharomyces cerevisiae. Mcd1 partially regulated the progression of meiotic recombination, sister chromatid separation, and nuclear division. DNA physical analysis during meiotic recombination showed that Mcd1 induced double-strand breaks (DSBs) but negatively regulated homologous recombination during DSB repair; Mcd1 expression delayed post-DSB stages, leading to inefficiencies in the DSB-to-joint molecule (JM) transition and subsequent crossover formation. These findings indicate that meiotic cells undergo Mcd1-mediated DSB formation during prophase I, and that residual Mcd1 could regulate the progression of JM formation during meiotic recombination.

Microarray Analysis of Gene Expression in Rat Hippocampus of Maternal Social Separation Model (모성 및 사회성 분리 백서 모델의 해마에서 유전자 칩을 이용한 유전자 발현 연구)

  • Lee, Hee Jae;Son, Chang Hee;Kwak, Hyong Ryol;Lee, Sang-Hyun;Han, Yoon Hee;Kim, Soo Young;Park, Jong-Ik;Chun, Wanjoo;Kim, Sung-Soo
    • Korean Journal of Biological Psychiatry
    • /
    • v.13 no.2
    • /
    • pp.110-116
    • /
    • 2006
  • Objectives : Alteration of hippocampus was demonstrated in the maternal social separation(MSS) pups, separated from dams on postnatal day(pnd) 14 and placed alone. Therefore, to understand the molecular events involved in the MSS, we have initiated a search for gene profiles that are up or down-regulated in the hippocampus of MSS pups. Methods : Analysis of cDNA microarray was performed by using total RNA extracted from the hippocampus of control and MSS pups on pnd 17. Also, passive-avoidance test was demonstrated on pnd 35. Results : Up-regulation of Nedd4a was observed in the hippocampus of MSS pups. Also, MSS rats showed less elongation of latency in passive avoidance test. Conclusion : We suggest that environmental effects of MSS may be altered the neural and/or glial differentiation and synapse formation-related genes which may lead cognitive alterations in MSS rats.

  • PDF

Electrophoretic Karyotypes of Fusarium oxysporum f. sp. lycopersici (Fusarium oxysporum f. sp. lycopersici의 Electrophoretic Karyotype)

  • Kim, Young-Tae;Kim, Hong-Gi
    • The Korean Journal of Mycology
    • /
    • v.27 no.2 s.89
    • /
    • pp.112-118
    • /
    • 1999
  • Strains of Fusarium oxysporum f. sp. lycopersici isolated from Korea, Japan and U.S.A. were used for electrophoretic karyotype (EK) analysis. Chromosome separations on FastLane agarose gels (FMC BioProducts, Rockland, ME), called pulsed field gel electrophoresis (PFGE), were performed by CHEF-DRII apparatus (Bio-Rad Laboratories, Melville, NY) using TAE as a running buffer. To obtain optimal condition for separation of chromosome sized DNAs, variable running conditions such as field strengths, swithching intervals, and running time were applied in CHEF gel electrophoresis. We were able to resolve 9 to 11 chromosome sized DNAs ranging in size from 0.76 to 6.41 Mb in isolates from Korea and estimate that the total genome size was ranging from 35.29 to 38.92 Mb. Distinct differences in length range and genome size exist among isolates from different countries. Isolates from Japan and U.S.A. were resolved 9 to 11 chromosome sized DNAs ranging in size from 1.24 to 6.85 Mb and estimated that the total genome size was ranging from 35.32 to 43.87 Mb. Isolates from variable provinces in Korea had the same or similar chromosomal polymorphism and showed different chromosomal DNA patterns compared to isolates from the other countries.

  • PDF

Expression of Polyhistidine-Containing Fusion Human HepG2 Type Glucose Transport Protein in Spodoptera Cells and Its Purification Using a Metal Affinity Chromatography

  • Lee, Chong-Kee
    • Biomedical Science Letters
    • /
    • v.16 no.3
    • /
    • pp.201-206
    • /
    • 2010
  • In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

Practical Packaging Technology for Microfluidic Systems (미소유체시스템을 위한 실용적인 패키징 기술)

  • Lee, Hwan-Yong;Han, Song-I;Han, Ki-Ho
    • Transactions of the Korean Society of Mechanical Engineers B
    • /
    • v.34 no.3
    • /
    • pp.251-258
    • /
    • 2010
  • This paper presents the technology for the design, fabrication, and characterization of a microfluidic system interface (MSI); the purpose of this technology is to enable the integration of complex microfluidic systems. The MSI technology can be applied in a simple manner for realizing complex arrangements of microfluidic interconnects, integrated microvalves for fluid control, and optical windows for on-chip optical processes. A microfluidic system for the preparation of genetic samples was used as the test vehicle to prove the effectiveness of the MSI technology for packaging complex microfluidic systems with multiple functionalities. The miniaturized genetic sample preparation system comprised several functional compartments, including compartments for cell purification, cell separation, cell lysis, solid-phase DNA extraction, polymerase chain reaction, and capillary electrophoresis. Additionally, the functional operation of the solid-phase extraction and PCR thermocycling compartments was demonstrated by using the MSI.

Phylogenetic Analysis of the Corticiaceae Based on Gene Sequences of Nuclear 18S Ribosomal DNAs

  • Lee, Seung-Shin;Jung, Hack-Sung
    • Journal of Microbiology
    • /
    • v.35 no.4
    • /
    • pp.253-258
    • /
    • 1997
  • The nuclear 18S ribosomal RNA genes of seven corticioid species were sequenced. These sequences were analyzed and compared with those of 24 other species of the order Aphyllophorales and phylogenetic trees were constructed using parsimonious methods. Phylogenetic analyses showed that two species among examined members of the Corticiaceae, Resinicium bicolor and Thanatephorus praticola, are located distantly from the remaining six species. The separation of R. bicolor seems to be kphylogenetically significant because it has very unique cystidia. The independent lineage of T. practicola suggests that it is also phylogenetically distinct because it has unusual features like the homobasidium producing secondary spores and the spetal ultrastructure of pore cap. Furthermore, Auriscalpium vulgare, Bondarzewia berkeleyi, and Heterobasidion annosum from different families of the Aphyllophorales proved to be closely related to the species of the Corticiaceae. They all have amyloid spores and grouped with Aleyrodiscus amorphus, which is a member of the Corticiaceae. The amyloidity of spores seems to be an improtant character throughout the order of the Aphyllophorales.

  • PDF

Microencapsulation Methods for Delivery of Protein Drugs

  • Yoon Yeo;Namjin Baek;Park, Kinam
    • Biotechnology and Bioprocess Engineering:BBE
    • /
    • v.6 no.4
    • /
    • pp.212-230
    • /
    • 2001
  • Recent advances in recombinant DNA technology have resulted in development of many new protein drugs. Due to the unique properties of protein druges, they have to be delivered by parenteral injection Although delivery of protein drugs by other routes, such as pulmonary and nasal routes, has shown some promises, to date most protein drugs are administered by par-enteral routs. For long-term delivery of protein drugs by parenteral administration, they have been formulated into biodegradable microspheres. A number of microencapsulation methods have been developed, and the currently used microencapsulation methods are reviewed here, The microen-capsulation methods have been divided based on the method used. They are: solvent evapora-tion/extraction; phase separation (coacervation);spray drying; ionotropic gelation/polyelectrolyte complexation; interfacial polyumerization and supercritical fluid precipitation. Each method is de-scribed fro its applications, advantages, and limitations.

  • PDF

Effects of Surface Geometry on Polyelectrolyte Adsorption

  • Park, Young-G.;Kim, Key-Seek;Sung, Ki-Chun
    • Journal of the Korean Applied Science and Technology
    • /
    • v.17 no.2
    • /
    • pp.149-156
    • /
    • 2000
  • For the adsorption of polyelectrolyte at the surface of polyacrylamide gel particle, preferential adsorption of the large polyelectrolyte such as DNA is governed by the surface area of an adsorbent. The adsorption equilibrium constant can be varied by surface geometry of porous polymer, and it can be described as a function of ionic strength and surface area. Physical parameters affecting the adsorption were estimated using the theoretical governing equation of polyelectrolyte which electrophoretically moved along the column, and geometrical surface area was estimated by Waldman-Mayer's physical model. The separation of polyelectrolytes was studied using the physical parameters estimated by ionic strength and surface geometry.