ABSTRACT
Previous methods used for nuclear transplantation were further investigated to develop a method that was both easy to carryout and did not require any special apparatus, such as Piezoimpact or Spindle-View. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by the direct injection of the donor nucleus or cell without pulling out the injection pipette. 200 oocytes were manipulated using this method and it cost about 40 seconds with nucleus injection and about 30 seconds with cell injection to complete a reconstructed embryo. The success rates were 62.6% and 86. 0%, respectively, and enucleation rate was about 73.3% validated by Hoechst 33342. Using this method, the nucleus was completely eliminated and another was injected using the microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice.
Subject(s)
Animals , Female , Male , Mice , Cell Culture Techniques , Methods , Cell Nucleus , Metabolism , Cells, Cultured , Cloning, Organism , Methods , Embryo, Mammalian , Cell Biology , Metabolism , Embryonic Development , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred Strains , Nuclear Transfer Techniques , Oocytes , Cell Biology , Metabolism , Zona Pellucida , MetabolismABSTRACT
We synthesized a novel (18)F-labeled dopamine D(4) receptor antagonist (Ki=4.3 nM), 3-(4-[(18)F]fluorobenzyl)-8-methoxy-1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one ([(18)F]FMTP), which has exhibited high affinity and selectivity. Radiosyntheses were accomplished by the reaction of fluorine-18-labeled intermediate with 8-methoxy-1,2,3,4-tetrahydrochromeno[3,4-c]pyridin-5-one (1) followed by HPLC purification. The overall radiochemical yield of the radiosynthesis was 19.5% (decay corrected), the specific radioactivity was about 110 GBq/micromol and the radiochemical purity was greater than 99%, the time of synthesis and purification was approximately 110 min. Tissue distribution studies of the [(18)F]FMTP in rats showed that the radioactivity in the brain was concentrated in frontal cortex and medulla, the region that has a high density of D(4) receptors. Pre-treatment with nonradioactive FMTP (1.0mg/kg) produced a significant reduction of radioactivity in all the regions. About 40% of total radioactivity in plasma and 100% in rat brain extract represented unchanged radioligand at 60 min after injection as determined by HPLC. These results indicate that [(18)F]FMTP have some specific binding to the D(4) receptor.
