By Aria Baniahmad
A panel of exceptional investigators surveys and explains the main state of the art equipment utilized in thryroid receptor (TR) examine and explains their useful experimental info. defined in step by step aspect to make sure powerful experimental effects, the suggestions offered disguise a large choice of key parts, together with TR in improvement and knockout (mouse and Xenopus), transcriptional law through TRs in either cell-free platforms and in residing cells, and TR mutant research of sufferers. extra equipment supply strong instruments for the isolation of TR-regulated protein complexes, for learning the oncogene v-Erba in blood telephone differentiation, and for aim gene research within the mind. Microarray chip equipment also are offered for interpreting the organs of transgenic mice to spot aim genes within the liver.
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Extra info for Thyroid Hormone Receptors: Methods and Protocols (Methods in Molecular Biology, Vol 202)
3. Section at 5 µm. 4. Stain with hemotoxylin-eosin by American Histolab (Rockville, MD). 5. To detect proliferating hepatocytes, inject mice intraperitoneally with BrdU, 50 mg per kg of body weight (Boeringer-Mannhein). 6. After 1 h, kill the mice immediately, and fix liver biopsies in 10% neutral-buffered formalin solution (Sigma). 7. Carry out immunostaining of BrdU and use TUNEL analysis by Molecular Histology Laboratories (Rockville, MD). 4. Notes 1. Control mice are injected with the same volume of PBS alone for 6 h before sacrifice and liver harvest.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Trypsinize ES cells. Inactivate the trypsin with normal ES medium and spin for 5 min at 800g. Wash the pellet twice with GMEM or OptiMEM. Count the washed cells. Mix 5 × 106 cells with 40 µg of the linearized recombination vector in a total volume of 800 µL GMEM or OptiMEM. Transfer the mixture into a 4-mm electroporation cuvette and perform the electroporation at 260 V and 500 µF. Wait for 20 min before seeding these cells on 5 B100 plates on a MEF layer resistant to the antibiotic used for the positive selection and add 8 × 10 5 nonelectroporated ES cells per plate.
However, this strategy is inefficient if the transcription of the targeted gene is too low in ES cells, since it will result in weak resistance of the recombined ES cells to G418 and obviate efficient selection. 3. It is very important to wait until the colony is large enough before cloning them. Look carefully at your plates during the selection. It sometimes happens that colonies stop growing in diameter and grow in thickness. The cells at the periphery then start to differentiate, and the clone will never cover the expected surface.