The following protocol is modified from the protocol that accompanies the Ecdysone-Induced Mammalian Expression System (Invitrogen); a similar system is sold by Stratagene. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.

Operation method

Ecdysone regulation induces cellular gene expression experiments

Materials and Instruments

Plasmid with luciferase reporter gene and expression induced by ecdysone Plasmid with gene of interest and expression induced by ecdysone pVgRXR Cultured mammalian cells
Neomycin Pine Steroid A Zeocin culture medium

Move

makings

Buffers and solutions
Dilute the storage solution to the appropriate concentration.

Neomycin

PINOSTERONE A
Another analog of ecdysone is muristerone A (Sigma Gongtwo).

Zeocin

Cultures

Culture medium suitable for the growth of cells to be transfected
Depending on the needs, the culture medium must be supplemented with Zeocin, Neomycin and Pine Sterone A.

Additional reagents

Steps 1, 2 and 6 of this protocol require the reagents listed in Chapter 16 Suitable Transfection Protocols.
Step 4 of this protocol requires the reagents listed in Scheme 6 of this chapter.
Step 8 of this protocol requires reagents listed in Chapter 6, Protocol 8, Chapter 7, or Appendix 8.

Vectors and Strains

Plasmids with a luciferase reporter gene and ecdysone-induced expression (e.g., Invitrogen pIND series)
Plasmids with a gene of interest and expression induced by ecdysone
pVgRXR (Invitrogen)

Cells and tissues

Cultured mammalian cells

Methods

1. Stabilize transfected cells with pVgRXR, using a transfection method appropriate for the cells under study (see Chapter 16 for selection of transfection protocols).
If pVgRXR is used, screen cells with this plasmid using Zeocip. For other plasmids, use a suitable antibiotic to obtain stable integrin colonies.


2. To select clones that selectively induce expression of the gene in the presence of ecdysone or its analogs, transiently transfect Zeocin-resistant colonies obtained in step 1 (see Chapter 16 for selection of transfection protocols) with a plasmid that carries a luciferase reporter gene and is subject to ecdysone-induced expression.

3. 24~96 h after transfection, replace the culture medium with fresh culture medium containing Zeocin, neomycin and 5umol/L pinostrobin A to induce luciferase expression. The cells were incubated at 37°C for 20 h in a humidified incubator with 5%~7% _CO2.
In order to optimize the induction of the expression of the reporter molecules, the amount of pinostrobin A and the duration of induction varied among different cell lines. To optimize the conditions, attempts were made in the range of 0.1-1umol/L of pinasterone A concentration and 16-72 h induction time.

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