High efficiency gene transfer and expression in mammalian cells by a multiple transfection procedure fo mar sequences
AssigneeSelexis S.A.
InventorsNicolas Mermod, Pierre Alain Girod, Philipp Bucher, Duc-Quang Nguyen, David Calabrese, Damien Saugy, Stefania Puttini
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Abstract
The present invention relates to purified and isolated DNA sequences having protein production increasing activity and more specifically to the use of matrix attachment regions (MARs) for increasing protein production activity in a eukaryotic cell. Also disclosed is a method for the identification of said active regions, in particular MAR nucleotide sequences, and the use of these characterized active MAR sequences in a new multiple transfection method.
Claims45
A purified and isolated DNA sequence comprising
- a) at least one bent DNA element,
- b) and at least one binding site for a DNA binding protein, wherein said DNA sequence has protein production increasing activity.
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A purified and isolated cLysMAR element and/or fragment having protein production increasing activity, a sequence complementary thereof, a part thereof sharing at least 70% nucleotides in length, a molecular chimera thereof, a combination thereof or variants.
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A synthetic MAR sequence comprising natural MAR elements and/or fragments assembled between linker sequences.
A method for identifying a MAR sequence using a Bioinformatic tool comprising computing values of one or more DNA sequence features corresponding to DNA bending, major groove depth and minor groove width potentials, and melting temperature.
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The method of claim 20 wherein the DNA binding protein is SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C/EBP, CREBP1, FOX, Freac7, HFH1, HNF3alpha, Nkx25, POU3F2, Pit1, TTF1, XFD1, AR, C/EBPgamma, Cdc5, FOXD3, HFH3, HNF3 beta, MRF2, Oct1, POU6F1, SRF, V$MTATA_B, XFD2, Bach2, CDP CR3, Cdx2, FOXJ2, HFL, HP1, Myc, PBX, Pax3, TEF, VBP, XFD3, Brn2, COMP1, Evil, FOXP3, GATA4, HFN1, Lhx3, NKX3A, POU1 F1 , Pax6, TFIIA or Vmw65 or a combination of two or more of these transcription factors.
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A method for identifying a MAR comprising providing at least one filter detecting clusters of DNA binding sites, wherein said filter detects said clusters using profiles or weightmatrices.
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A method for transfecting a eukaryotic host cell, said method comprising a) introducing into said eukaryotic host cell at least one purified DNA sequence comprising at least one DNA sequence of interest and/or at least one purified and isolated DNA sequence consisting of a MAR nucleotide sequence or other chromatin modifying elements, b) subjecting within a defined time said transfected eukaryotic host cell to at least one additional transfection step with at least one purified DNA sequence comprising at least one DNA sequence of interest and/or with at least one purified and isolated DNA sequence consisting of a MAR nucleotide sequence or other chromatin modifying elements c) selecting said transfected eukaryotic host cell.
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A transfected eukaryotic host cell comprising at least one purified DNA sequence comprising at least one DNA sequence of interest and/or at least one purified and isolated DNA sequence consisting of a MAR nucleotide sequence or other chromatin modifying elements.
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Dependencies are inferred from the claim text.