Construct and Sequence for Enhanced Gene Expression

Abstract

The invention relates to a method for transcription and expression using a nucleic acid construct which is characterized by the presence of a promoter followed by an intronic promoter. The invention further relates to said nucleic acid construct, an expression vector and a cell comprising said construct, and its use. The invention also relates to methods for transcription and optionally expression using a nucleotide sequence. The invention further relates to said nucleotide sequence and a construct, expression vector and cell comprising said nucleotide sequence, and its use.

1 .- 15 . (canceled) 
     
     
         16 . A nucleic acid construct comprising a first promoter, a second promoter, and a single nucleotide sequence of interest, wherein said first promoter and second promoter are constitutive promoters and are both operably linked to said single nucleotide sequence of interest, and wherein said second promoter is an intronic promoter flanked by a first intronic sequence located upstream of said second promoter and a second intronic sequence located downstream of said second promoter, and wherein said single nucleotide sequence of interest is under the control of said first promoter and said second promoter. 
     
     
         17 . The nucleic acid construct according to claim  1 , wherein said nucleic acid construct further comprises an additional expression regulating sequence, and wherein said additional expression regulating sequence, said first promoter and said second promoter are all operably linked to said nucleic acid sequence of interest. 
     
     
         18 . The nucleic acid construct according to claim  1 , wherein said nucleotide sequence of interest encodes a protein or polypeptide of interest. 
     
     
         19 . An expression vector comprising the nucleic acid construct according to claim  1 . 
     
     
         20 . An in vitro cell comprising the nucleic acid construct according to claim  1 . 
     
     
         21 . A non-human cell comprising the nucleic acid construct according to claim  1 . 
     
     
         22 . The nucleic acid construct according to claim  2 , wherein said additional expression regulating sequence comprises or consists of an intron. 
     
     
         23 . The nucleic acid construct according to claim  3 , wherein said protein or polypeptide of interest is a heterologous protein or polypeptide. 
     
     
         24 . The nucleic acid construct according to claim  1 , wherein said first promoter comprises a sequence having at least 50% identity to SEQ ID NO: 1 over its whole length or having at least 50% identity to SEQ ID NO: 2 over its whole length. 
     
     
         25 . The nucleic acid construct according to claim  1 , wherein said second promoter comprises a sequence having at least 95% identity to SEQ ID NO: 57, 58, or 79 over its whole length. 
     
     
         26 . The nucleic acid construct according to claim  1 , wherein said second promoter comprises a sequence having at least 95% identity to SEQ ID NO: 57 or 58 over its whole length. 
     
     
         27 . The nucleic acid construct according to claim  1 , wherein said first promoter comprises a sequence having at least 50% identity to SEQ ID NO: 1 or 2 over its whole length and said second promoter comprises a sequence having at least 95% identity to SEQ ID NO: 57, 58 or 79 over its whole length. 
     
     
         28 . The nucleic acid construct according to claim  1 , wherein said first promoter comprises a sequence having at least 50% identity to SEQ ID NO: 1 over its whole length and said second promoter comprises a sequence having at least 95% identity to SEQ ID NO: 57 or 58 over its whole length. 
     
     
         29 . The nucleic acid construct according to claim  1 , wherein said first promoter comprises a sequence having at least 50% identity to SEQ ID NO: 2 over its whole length and said second promoter comprises a sequence having at least 95% identity to SEQ ID NO: 57 or 58 over its whole length. 
     
     
         30 . The nucleic acid construct according to claim  1 , wherein said second promoter is a human or murine cytomegalovirus (CMV) promoter. 
     
     
         31 . The nucleic acid construct according to claim  1 , wherein said first promoter and said first intronic sequence comprise the sequence of SEQ ID NO: 1. 
     
     
         32 . The nucleic acid construct according to claim  1 , wherein said second intronic sequence comprises the sequence of SEQ ID NO:19. 
     
     
         33 . A method for transcription and optionally purifying the produced transcript comprising the steps of:
 a) providing a nucleic acid construct comprising a first promoter, a second promoter, and a single nucleotide sequence of interest, wherein said first promoter and second promoter are constitutive promoters and are both operably linked to said single nucleotide sequence of interest, and wherein said second promoter is an intronic promoter flanked by a first intronic sequence located upstream of said second promoter and a second intronic sequence located downstream of said second promoter, and wherein said single nucleotide sequence of interest is under the control of said first promoter and said second promoter,   b) contacting a cell with said nucleic acid construct to obtain a transformed cell; and,   c) allowing said transformed cell to produce a transcript of the nucleotide sequence of interest; and optionally,   d) purifying said produced transcript.   
     
     
         34 . A method for transcription and optionally purifying the produced transcript comprising the steps of:
 a) providing a nucleic acid construct comprising in the 5′ to 3′ direction an expression enhancing element, a heterologous promoter and a nucleotide sequence of interest, wherein said expression enhancing element has at least 50% identity to SEQ ID NO: 1 or 2 over its whole length, and wherein said expression enhancing element and said heterologous promoter are operably linked to a same, single nucleotide sequence of interest; and,   b) contacting a cell with said nucleic acid construct to obtain a transformed cell; and,   c) allowing said transformed cell to produce a transcript of the nucleotide sequence of interest; and optionally,   d) purifying said produced transcript.