[n order to obtain the trlle expected DNA prod uct from PCR and RT-PCR using genornic DNA or cDNA reversely transcribed from mRNA. the PCR should be done in an appropriated condition. Sometimes the PCR was repeatedly fail ed. and cventllally the PCR product was turned out to be nonspecific and rudimentary . And more‘ t he PCR prodllctwas not reproducible even though careflll repeat of experiments. As the PCR was based on the exact primel hybridization. the condition of primer hybridization should be properly controlled by a nnealing temperatllre. But the selection of primer seqllences for targeting a specific gene is mostly important. A new method of primer eval uation is now available llsing DNA base pair polarity program. This study presents an example of PCR targeting to human Bax gene using genomic DNA. The DNA base pair polarity theory can di vide the genetic cord into propel DNA segments and calclllaLe their DNA base pair hybridization energy. Thus. mathematically the degree 0(' exact primer hybridization can be expected for the t r1l8 targeting of PCR. However, the DNA base pair polal'ityanalysis demonstrates that the more frequent number of DNA segment incl'eased the specificity of PCR. but decreased its sensitivity . While the greater polarity of DNA segment composed of increased nllmber of polarized DNA base pairs showed increased sensitivi ty 0 1' PCR. bllt relati vely decreased specificity of PCR. With the mllltiple analysis of PCR. especially for PCR cloning from the gDNA and cDNA, we found that the primers themselves showed secondary strllcture of partial hybridization between sameprimers or each pair primers. The DNA base pail‘ polarity signal can directly demonstrated symmetric sequences 0 1' each primer. and also can distinguish the dimmer formation from each pair primers. At least the symmetric seqllence of fOlll‘ base pairs dramatically showed the dimrner formation. On the other hand. in addi tion Lo the statlls of DNA base pair polarity the three-dimensional strllctllre of DNA dOllble helix targeted by the primer seqllences may affect the sensitivity and specificity of PCR detection. The present study introduced a new method of primer evalllation and selection in order to obtain abundant and exacL! y-trlle DNA product for genomic ffilltation analysis and gene expression profï le

About 10 percent of quasars are known to exhibit deep broad absorption troughs blueward of prominent permitted emission lines, which are usually attributed to the existence of outflows slightly above he accretion disk around the supermassive black hole. Typical widths up to 0.2c of these absorption roughs indicate the velocity scales in which special relativistic effects may not be negligible. Under he assumption of the ubiquity of the broad absorption line region in quasars, the broad emission line flux will exhibit Thomson scattered components from these fast outflows. In this paper, we provide our Monte Carlo calculation of linear polarization of singly Thomson scattered line radiation with the careful considerations of special relativistic effects. The scattering region is approximated by a collection of rings that are moving outward with speeds υ =cβ < 0.2c near the equatorial plane, and the scattered line photons are collected according to its direction and wavelength in the observer's rest frame. We find that the significantly extended red tail appears in the scattered radiation. We also find that the linear degree of polarization of singly Thomson scattered line radiation is wavelength-dependent and hat there are significant differences in the linear degree of polarization from that computed from classical physics in the far red tail. We propose that the semi-forbidden broad emission line C III]1909 may be significantly contributed from Thomson scattering because this line has small resonance scattering optical depth in the broad absorption line region, which leads to distinct and significant polarized flux in this broad emission line.

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

The aim of this study was to investigate whether addition of porcine epididymal fluid (pEF) into culture medium during in vitro maturation influences the nuclear maturation of porcine germinal vesicle (GV) oocytes. Porcine cumulus-oocyte complexes (COCs) from follicles were cultured in tissue culture medium 199 (TCM 199) containing pEF. After 48 hr of culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. The proportion of oocytes reaching at metaphase II (M II) stage was significantly (p<0.05) increased in oocytes cultured in the media supplemented with 10% pEF during in vitro maturation than in those without pEF regardless of cumulus presence or absence (54.6% vs 22.5%，51.7% vs 24.2%). The supplementation of pEF during maturation of oocyte enhanced oocytes maturation in a dose-dependent manner in vitro. Also significant differences (p<0.05) in the percentage of MII oocytes were observed according to exposure period in pEF. Present study suggests that pEF contains a enhancing component(s) for nuclear maturation of porcine immature oocytes in vitro.

Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including microdroplet (MD), openpulled straw (OPS) and electron microscopic grid (EMgrid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the postvitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RTPCR and immunofluorescence assays. The survival rates of the postvitrified human ES cells using MD, OPS and EMgrid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slowfreezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slowfreezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.

To study the signaling effect of insulin-like growth factor-Ⅰ(IGF-1), transgenic mice containing IGF-1 Receptor (IGF-1R) cDNA fused to metallothionein promoter were produced by DNA microinjection into the pronucleus of mouse zygote. Three founders were produced with transgenic mice containing IGF-1R gene. Transgenic mice lines contained approximately 4～20 copies of transgenes per cell and transmission of this gene into the progeny with Mendelian manner were determined. The founder mice were mated with normal mice to produce F1 mice and then F2 mice. Transmission rates of IGF-1R transgene in the progeny mice were 25～60% in F1 generation and 40～50% in F2 generation. The mRNA expression of IGF-1R transgene in liver was analyzed using RT-PCR for IGF-1R gene in liver. When body weights of transgenic pups were measured during 4, 10 and 14 weeks after birth, IGF-1R transgenic mice grew faster than non transgenic littermates. This study indicated that growth regulation by IGF-1 signaling through IGF-1R can be elucidated using IGF-1R transgenic mice.