Patent ID: 8680256

Claim:
A method for producing inducible expression active linear gene constructs, comprising the step of: (a) generating an inducible expression active linear gene construct, comprising one or more control element(s) that are inducible element(s), a minimal promoter comprising a transcriptionally non-inducible constitutively active ribosomal protein gene promoter, a DNA sequence selected from a gene, a coding region, an open reading frame (ORF), an inhibitory RNA coding sequence and a cDNA, a 3′ untranslated region (3′ UTR) containing mRNA destabilization or stabilization elements of a 3′ UTR of a cellular mRNA, and a termination sequence, wherein the generating step comprises a PCR amplification of a source expression polynucleotide comprising in 5′ to 3′ direction a promoter sequence and the DNA sequence using (i) a forward primer comprising at its 3′ end, a first sequence part complementary to a promoter region of the source expression polynucleotide upstream of the DNA sequence, and at its 5′ end a second sequence part comprising one or more introduced control element(s); and (ii) a reverse primer selected from a reverse primer complementary to a region of the source expression polynucleotide downstream of the DNA sequence, wherein the primer comprises mRNA destabilization or stabilization elements of a 3′ UTR of a cellular mRNA and a termination sequence, or a reverse primer complementary to a region of the source expression polynucleotide downstream of a 3′ UTR wherein the region contains mRNA destabilization or stabilization elements of a 3′ UTR of a cellular mRNA, wherein the source expression polynucleotide is a vector, a lentivirus, a plasmid, a virus-based vector, or a linear or linearized or amplified fragment thereof, wherein the mRNA destabilization elements are selected from AU-rich elements, GU-rich elements, and U-rich sequences, wherein the mRNA stabilization elements are selected from GC-rich elements, CU-rich elements, and UG-rich sequences, wherein the generating step does not involve a cloning step, wherein said cloning step includes the use of one or more of restriction digestion, cloning enzyme(s), bacterial transformation and plasmid preparation, and wherein expression of the inducible expression active linear gene construct resulting from (a) can be induced by the addition of a compound that activates the expression or by the withdrawal of a compound that represses the expression.