Xip Labus egg extracts are useful as cytoplasmic and organelle material for in vitro reconstitution of cellular events such as nuclear assembly and nuclear protein import. When sperm chromatin DNA is added to either crude or fractionated XUS egg extracts, membrane vesicles present in the extract, bind to the surface of the chromatin fuse, and incorporate nuclear pore components to form fully functional synthetic nuclei. Characterization of the molecular basis of these processes is then possible by immuno depletion of extracts and by protein edition.
Hi, my name's Marie Cross and I'm a graduate student in Dr.Maureen Power's lab in the Department of Cell Biology at Emory University in Atlanta, Georgia. Today we will show you how to form nuclei in vitro using fractionated zenap egg extracts. This procedure involves the following steps, performing the nuclear assembly reaction and assessing nuclei formation.
So let's get started. To begin nuclear assembly, take an aliquot of fractionated cytosol and add phosphocreatine from a 0.2 molar stock to a final concentration of 20 millimolar. Then add creatine kinase from a five milligram per milliliter stock to a final concentration of 50 micrograms per mil.
Finally, add 20 micromolar A TP using a 0.2 molar stock. Next, add light membranes to the cytosol at a one to 10 dilution. This approximates the amount of membrane present in the original crude extract.
Mix all the components by up and down pipetting to disperse the membranes in the extract. This must be done before the sperm chromatin is added. Next, add sperm chromatin to the fractionated extract at the desired concentration and immediately mix the extract and sperm together by gentle up and down pipetting with a cutoff tip.
When the sperm chromatin is added to the extract, it immediately begins to decon, condense, and become susceptible to shearing. Generally, these reactions are set up in 500 microliter centrifuge tubes with a total volume less than a hundred microliters. The egg extracts will support the formation of up to 8, 000 to 9, 000.
Nuclei per microliter incubate the mixture for one to two hours at room temperature to allow the nuclei to form. Nuclei are visualized using staining. After approximately 30 minutes, the newly formed nuclei will be small and oval in shape.
The DNA is still quite condensed and the nucleus should appear to be a solid light blue from hook spining when observed in the UV channel as the DNA condenses. Further, the blue staining will be less homogeneous with black gaps eventually appearing. Nuclear membrane formation can be visualized by adding one microgram per male of three three diaa carbox amine or DHCC in the fixative.
DHCC is a fluorescent lipophilic dye that is visualized in the fluorescein channel. In the early stages of nuclei, assembly, membrane vesicles are bound on the chromatin surface and begin to fuse in patches. This results in an uneven and discontinuous appearance of the forming membrane.
Once the membrane is complete, it smooths out over the nuclear surface and will appear as an even unbroken rim around the DNA. There will be a fair amount of background fluorescence in the surrounding cytosol due to membrane vesicles, which have not incorporated into nuclei. We've just shown you in vitro nuclear reconstitution using soluble and light membrane fractions from xenopus egg extract.
It's important to remember that crude extract can be used for nuclear assembly, but not after freezing and thawing. So that's it. Thanks for watching and good luck with your experiments.
