An improved double Recoverable Block of Function (RBF) system for
transgene containment with enhanced reliability is disclosed. The system
includes two blocking constructs (BCs) at the opposite sides of a
transgene of interest (TGI). The system comprises means for recovery of
the function blocked by expression of the blocking genes of the BCs. The
invention is also related to a kit comprising a cloning vector having the
BCs and TGI or alternatively a place for TGI to be inserted in.
Segregating (two-insert) and inducible (one-insert) types of double RBF
are disclosed. The two BCs are either similar or different in their DNA
and RNA structures. The two BCs may encode same protein but promoters
driving the blocking genes are different and/or the coding sequence of
the blocking genes is modified by codon preference change. A system
comprising two BCs having different blocking genes is also disclosed. The
system may consist of one or two Recovering constructs (RC), but also a
system recovering without an RC is disclosed. Whether the system includes
an RC or not, the recovery is induced by an external intervention that is
controlled by human being.