BD RevTet™ System使用說明

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 BD RevTet™ System使用說明

pRevTet-On ，pRevTet-Off ，pRevTet-Off-IN， pRevTRE 

 Introduction

The BD RevTet-OnTM & BD RevTet-OffTM Systems combine the advantages of

retrovirus-mediated gene transfer with the proven tetracycline-regulated control

of BD Tet-OnTM and BD Tet-OffTM Gene Expression Systems. BD RevTetTM allows

the fast and efficient establishment of regulated gene expression systems in a

wide variety of cell types.

Each RevTet System is a complete retroviral gene expression system containing

a retroviral regulator （r）tTA vector, a retroviral response vector, a control vector,

and a packaging cell line. Like our BD Retro-XTM System （#631508）, the RevTet

Systems contain the BD RetroPackTM PT67 Packaging Cell Line （#631510） for

the safe, efficient production of infectious, replication-incompetent retrovirus. The

RevTet Systems provide a powerful and convenient approach to setting up the

tetracycline （Tc）-inducible, high-level gene expression systems first described

by Gossen and Bujard （1992）.

Retroviral-mediated tet-regulation

The Tet Systems are based on two elements from the tet operon of the E. coli

Tn10 transposon—the Tet repressor protein （TetR） and the tet operator DNA

sequence （tetO）. The gene to be expressed is cloned into the pRevTRE response

vector downstream of the tetracycline-responsive element （TRE）, which contains seven direct repeats of the 42-bp tetO sequence and the minimal immediate early promoter of cytomegalovirus （P

minCMV）. The two systems differ in their

regulatory vector （Figure 1）. The RevTet-Off System uses the pRevTet-OffVector, which contains the tTA regulatory element, a fusion of TetR and the VP16

activation domain. The BD RevTet-OnTM System uses pRevTet-On, based on the

reverse tTA （rtTA）. Both systems use the pRevTRE response vector. All the RevTet

Vectors are derived from pLNCX, a BD Retro-XTM vector capable of producing

high-titer virus in the appropriate packaging cell lines.

In the RevTet-Off System, tTA binds the TRE and activates transcription in the

absence of Tc or the Tc derivative doxycycline （Dox）. Thus, as Tc or Dox is added

to the culture medium, transcription is turned off in a dose-dependent manner

（Figure 1）. Conversely, the RevTet-On System allows gene expression to be activated by the addition of Dox. These two complementary systems allow you to

choose the one that best suits your needs.

Benefits and applications of Tc-mediated induction

In most inducible mammalian gene expression systems （e.g., induction by heavy

metals, steroid hormones, or heat shock）, induction is nonspecific and expression levels cannot be precisely regulated. In addition, these systems are generally leaky in the “off” state, and the inducing agent itself may be cytotoxic or have

pleiotropic effects on results. In contrast, regulation of gene expression by the

heterologous bacterial control elements in the RevTet Systems is very specific.

Furthermore, the levels of Tc or Dox required for the full range of gene expression are not cytotoxic and have no significant effect on cell proliferation or animal

growth, even with continuous treatment （Bohl et al., 1997; Mayford et al., 1996）.

The use of separate regulatory and response vectors in the RevTet Systems

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