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  • 1
    Electronic Resource
    Electronic Resource
    Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons (1993)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 14 (1993), S. 296-304 
    Details
    ISSN: 0192-253X
    Keywords: Transport ; nicotinic acetylcholine receptor ; sympathetic neurons ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations ≥ 20 μM. This temperature sensitive, saturable component is effectively competed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotides described in non-neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro.Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit α3; nAChR α3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by =80%-90%. Furthermore, following antisense deletion of α3, “mutant” nAChRs with distinct functional characteristics are expressed.In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons. In addition, the functional studies emphasize the need for sensitive and specific assay following antisense deletion, since other homologous gene products may substitute for the targeted gene resulting in new phenotypes that are subtly different from wild type. © 1993Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020140407
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  • 2
    Electronic Resource
    Electronic Resource
    A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 33 (1992), S. 489-491 
    Details
    ISSN: 1040-452X
    Keywords: Oocytes ; Cattle ; Sperm ; Fertility ; Microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as “tail stump defect.” No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasma. SPZ were treated with 0.1 μM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at - 25°C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39°C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively. © 1992 Wiley-Liss, Inc.
    Additional Material: 1 Tab.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080330416
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  • 3
    Electronic Resource
    Electronic Resource
    Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 36 (1993), S. 130-138 
    Details
    ISSN: 1040-452X
    Keywords: Chimera ; Micromanipulation ; Pluripotency ; ICM ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080360203
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  • 4
    Electronic Resource
    Electronic Resource
    Potential of hypertonic medium treatment for embryo micromanipulation: I. Survival of rabbit embryos in vitro and in vivo following sucrose treatment (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 27 (1990), S. 110-117 
    Details
    ISSN: 1040-452X
    Keywords: Rabbit embryos ; Hypertonic sucrose ; Micromanipulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Rabbit zygotes and embryos were exposed to hypertonic sucrose in phosphate-buffered saline (SPBS). In experiment one, 144 zygotes shrank to 32-36% of their initial volume in 1.0 M SPBS within 30 min. Neither hypertonic treatment with 0.5 M or 1.0 M SPBS nor micropuncture of the zona pellucida after shrinkage affected embryo development into blastocysts in vitro (88%, 83%, and 82%, respectively), compared to that of the controls (93%, P 〉 .05). In experiment two, 252 two- to four-cell- and 177 morula-stage embryos were exposed to isotonic PBS control or 0.5 M, 1.0 M, or 1.5 M SPBS for 30, 60, 90, 120, and 150 min before transfer to PBS (290 mOsm). Embryo development was significantly reduced (P 〈 .05) when embryos were exposed in 0.5 M and 1.0 M SPBS for more than 60 min or in 1.5 M SPBS for more than 30 min. In experiment 3, morulae exposed for 60 min to 0.5 M or 1.0 M SPBS shrank to 37-39% or 32-35% of their initial volume and then expanded to 87-94% or 81-90% of their initial volume, respectively, after being returned to isotonic PBS for 60 min, but embryos in 1.5 M SPBS had erratic osmotic behavior. In experiment four, 192 two- to four-cell embryos exposed to 0.5 M SPBS for 0, 30, and 60 min before transfer to oviducts of recipients resulted in the production of 39%, 42% and 31% young, respectively (P 〉 .05). Exposure of embryos to 0.5 M sucrose for 60 min clearly does not compromise developmental potential and can simplify and speed up micromanipulation procedures.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080270205
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  • 5
    Electronic Resource
    Electronic Resource
    Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 27 (1990), S. 118-129 
    Details
    ISSN: 1040-452X
    Keywords: Rabbit embryos ; Micromanipulation ; Nuclear transfer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca++-free PBS for 15 min at 39°C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 μm or 25 μm at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P 〉 .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080270206
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  • 6
    Electronic Resource
    Electronic Resource
    Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen α1, lysyl oxidase, and cyclooxygenase-1 genes in human embryo lung fibroblasts (1998)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 71 (1998), S. 254-263 
    Details
    ISSN: 0730-2312
    Keywords: lysyl oxidase ; cyclooxygenase-1 ; type I collagen α1 ; prostaglandin E2 ; prostaglandin E2 receptors ; cyclic AMP ; interleukin-1β ; transforming growth factor-β ; forskolin ; 11-deoxy PGE1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin-1β( (IL-1β) and transforming growth factor-β (TGFβ) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase-1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 1996]. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11-deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11-deoxy PGE1 decreases basal and TGF-β induced type I collagen α1 (COL) mRNA, basal and IL-1β induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2or 11-deoxy PGE1. It suppresses both basal and TGF-β induced COL mRNA levels. Both PGE2 and 11-deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat-1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat-1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP. J. Cell. Biochem. 71:254-263, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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