By Martin J. Tymms, Ismail Kola
Hugely expert investigators with vast event in gene focusing on and mouse genetics describe their most sensible ideas for the layout of focusing on constructs and for the research of the mouse phenotype. those comprise embryo transplantation, in vitro embryonic stem cellphone differentiation, construction of aggregation chimeras, mouse pathology, embryo cryopreservation, and transplantation. cutting-edge and hugely sensible, Gene Knockout Protocols not just constitutes a useful resource of without problems reproducible suggestions for these simply getting into the sphere of gene focusing on, but in addition a key reference for all genetic researchers at the present time.
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Additional info for Gene Knockout Protocols
8, a conditional gene targeting strategy would be to induce the expression of the Cre gene under the control of exogenously added muristerone A by combining the genetic elements from several independent mouse lines carrying (1) a transgene using a promoter to express an EcR-IRES-RxR gene cassette, (2) a transgene consisting of the muristerone A-responsive promoter to express the Cre gene, and (3) a conditionally targeted locus with the loxP site flanking the endogenous sequences to be deleted. 4.
13. 5 mL of freezing medium. 14. Mix gently, aliquot into five cryopreservation vials, and store overnight at –80°C in a partially insulated box so that the cells freeze slowly. The next day transfer to liquid N2. 3. 1 Electroporation Day 0 1. Split one plate of subconfluent ES cells into four dishes such that the next day the cells are ready to be split again (Fig. 2A) (see Note 6). Day 1 2. Change the media in the morning, as these cells are plated more confluently than usual (Fig. 2B). 3. In the afternoon, trypsinize plates as previously described and collect the cells.
Place uterine horns into a 10-cm tissue culture dish with PBS (in tissue culture hood) and dissect out embryos. 5. Place embryos in a fresh dish with 10 mL PBS. Decapitate and eviscerate the embryos. 6. Place carcasses into a clean 10 cm dish and mince them with a sterile razor blade until a gelatinous mass is created. 7. Add 5 mL of DMEM and transfer the mix into a 200-mL flask containing a stirbar. 8. Add 50 mL of MEF digestion media. 9. Stir at ) 37°C on a warm plate for 30–40 min. 10. Pipet off supernatant and save in a 50-mL tube.