WO2011153440A2 - Système de mutagenèse par transposons et ses méthodes d'utilisation - Google Patents

Système de mutagenèse par transposons et ses méthodes d'utilisation Download PDF

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Publication number
WO2011153440A2
WO2011153440A2 PCT/US2011/039080 US2011039080W WO2011153440A2 WO 2011153440 A2 WO2011153440 A2 WO 2011153440A2 US 2011039080 W US2011039080 W US 2011039080W WO 2011153440 A2 WO2011153440 A2 WO 2011153440A2
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cell
vector
transposase
transposon
transposons
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PCT/US2011/039080
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WO2011153440A3 (fr
WO2011153440A9 (fr
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Branden S Moriarity
Aaron M. Geurst
David A. Largaespada
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Regents Of The University Of Minnesota
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Publication of WO2011153440A9 publication Critical patent/WO2011153440A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host

Definitions

  • Retroviral mutagenesis is skewed by the natural predisposition of viral insertions into the 5' end or promoter regions of genes and it can also be hard to track insertion of retroviruses due to the large amount of native viral DNA in the host genome.
  • ENU mutagenesis is not easily trackable and requires genetic crosses, which are not even possible with cultured cells, to identify the location of mutations by this chemical mutagen.
  • Other methods include RNAi or cDNA overexpression library screens. RNAi can only give phenotypes by knocking down genes and thus cannot identify genes that need to be over-expressed to induce the phenotype of interest.
  • a method may include incubating a cell that mcludes at least 2 transposons and a polynucleotide encoding a transposase that causes transposition of the transposons to genomic DNA of the cell.
  • the incubating is under conditions suitable for growth and results in mutagenized cells having transposons that have transposed to genomic DNA.
  • the cell may be, for instance, a human cell or a murine cell, such as a mouse cell.
  • a vector is a replicating polynucleotide, such as a plasmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide.
  • the vector may include a coding sequence.
  • a vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector.
  • a vector can be both a cloning vector and an expression vector.
  • a vector may replicate in a eukaryotic cell.
  • a vector includes an origin of replication.
  • origins of replication are known in the art and are routinely used.
  • an origin is the OriP origin from an Epstein Barr virus.
  • OriP is a polynucleotide sequence to which an EBNA-1 polypeptide binds to result in replication of a polynucleotide containing the OriP polynucleotide.
  • the EBNA-1 polypeptide may be encoded by a coding region present in the vector and acting in cis, or may be encoded by a coding region not present in the vector and acting in trans.
  • a vector may include polynucleotides to permit replication in a prokaryotic cell and selection of prokaryotic cells containing a vector. Origins of replication and selectable markers useful for replication and selection in prokaryotic cells are known to the skilled person, are routinely used, and are readily available.
  • a nucleic acid sequence is "flanked by" cis-acting nucleotide sequences if at least one cis-acting nucleotide sequence is positioned 5' to the nucleic acid sequence, and at least one cis-acting nucleotide sequence is positioned 3' to the nucleic acid sequence.
  • a nucleic acid sequence flanked by cis-acting nucleotide sequences may be referred to herein as a "flanked sequence.”
  • Cis-acting nucleotide sequences include at least one inverted repeat at each end of the transposon, to which a transposase binds. Transposases is described in greater detail herein.
  • the disruptive sequence includes a splice acceptor (SA) site.
  • SA splice acceptor
  • a splice acceptor site is a nucleotide sequence that is generally involved in RNA splicing to remove intronic RNA sequences. While not intending to be bound by theory, the splice acceptor site is normally involved in the excision of introns, during which it is bound by an RNA-protein complex referred to as a spliceosome, cleaved, and then joined to a splice donor site that has already been cleaved, resulting in the excision of an mtervening portion of the nucleotide sequence in a lariat formation.
  • polynucleotide from a plasmid where the polynucleotide includes an OncX transposon, which contains, going from the 5' to the 3' end, a piggyBac ITR sequence, a Sleeping Beauty IR/DR sequence, a first SA site, a first pA site, a promoter (Cagg), an SD site, and (in inverted orientation), a second pA site and a second SA, flanked at the end by a Sleeping Beauty IR/DR sequence and a piggyBac ITR sequence, marking the end of the transposon.
  • OncX transposon which contains, going from the 5' to the 3' end, a piggyBac ITR sequence, a Sleeping Beauty IR/DR sequence, a first SA site, a first pA site, a promoter (Cagg), an SD site, and (in inverted orientation), a second pA site and a second SA, flanked at the end by a Sleep
  • a vector may include at least 1 transposon, at least 2 transposons, at least 3 transposons, at least 4 transposons, at least 5 transposons, at least 6 transposons, at least 7 transposons, at least 8 transposons, at least 9 transposons, at least 10 transposons, at least 11 transposons, or at least 12 transposons.
  • Transposons of the present invention preferably include an insertional mutagen which increases the ability of the transposon to induce a tumor or other phenotypic change upon insertion into a genomic coding sequence and/or regulatory sequence of a cell.
  • Alteration of the nucleic acid sequence will, in turn, affect the level of expression or the nature of the product expressed.
  • the nucleic acid is referred to as a disruptive sequence.
  • the nucleic acid is referred to as an affective sequence.
  • a disruptive sequence can induce various types of mutations, including, for example, C-terminal truncations, N- terminal truncations, and insertion of promoters and/or enhancers.
  • a tumor suppressor gene is a gene that reduces the probability that a cell will turn into a tumor cell. A mutation or deletion of such a gene will increase the probability of the cell containing the damaged gene to become a tumor cell. On the other hand, increased production of a tumor suppressor gene through, for example, increased promoter activity, can decrease tumor formation. Tumor suppressor genes also include growth suppressors, recessive oncogenes, and anti-oncogenes.
  • the location of the transposon can also be determined using a restriction endonuclease capable of cleaving a restriction site within the transposon. This yields at least one restriction fragment containing at least a portion of the integrated transposon, which portion includes at least a portion of an inverted repeat sequence along with an amount of genomic DNA of the cell that is adjacent to the inverted repeat sequence.
  • a restriction endonuclease capable of cleaving a restriction site within the transposon.
  • the specificities of numerous endonucleases are well known and can be found in a variety of publications, e.g. Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: New York (1989).
  • the polynucleotide of the transposon thus preferably includes a restriction endonuclease recognition site, preferably a 6-base recognition sequence.
  • the present invention also provides a kit for detecting a coding region involved in a phenotype.
  • the kit includes one or more of the vectors described herein in a suitable packaging material in an amount sufficient for transformation into cells to permit replication of the vectors.
  • other reagents such as buffers and solutions needed to practice the invention are also included.
  • Instructions for use of the packaged polypeptide or primer pair are also typically included.
  • pCEP4- 50ncX-PB7-ERt2 Figure 2d.
  • the transposase is tamoxifen inducible by virtue of fusing a mutated estrogen receptor to the PB7 Transposase.
  • the pCEP4-50ncX-PB7-ERt2 vector has dsRED and the Hygromycin resistance gene.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
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  • Biotechnology (AREA)
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  • Plant Pathology (AREA)
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  • Bioinformatics & Computational Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des méthodes utilisant des transposons pour modifier un phénotype d'une cellule in vitro. Une méthode peut consister à incuber une cellule comprenant au moins deux transposons et un polynucléotide codant pour une transposase à l'origine de la transposition des transposons à l'ADN génomique de la cellule; et à observer un phénotype dans les cellules mutagénisées obtenues. L'invention concerne également un système de mutagenèse par transposons qui permet d'introduire un polynucléotide dans l'ADN génomique d'une cellule in vitro.
PCT/US2011/039080 2010-06-04 2011-06-03 Système de mutagenèse par transposons et ses méthodes d'utilisation WO2011153440A2 (fr)

Applications Claiming Priority (2)

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US39695710P 2010-06-04 2010-06-04
US61/396,957 2010-06-04

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WO2011153440A2 true WO2011153440A2 (fr) 2011-12-08
WO2011153440A3 WO2011153440A3 (fr) 2012-03-22
WO2011153440A9 WO2011153440A9 (fr) 2012-12-27

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Citations (11)

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US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
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US20100221824A1 (en) 2000-10-31 2010-09-02 University Of Notre Dame Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggyBac
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Patent Citations (11)

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Publication number Priority date Publication date Assignee Title
WO1996001313A1 (fr) 1994-07-01 1996-01-18 Hermann Bujard Modulateurs de transcription regules par la tetracycline
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
WO1999025817A2 (fr) 1997-11-13 1999-05-27 Regents Of The University Of Minnesota Systeme de transposon a base d'adn permettant d'introduire un acide nucleique dans l'adn d'une cellule
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US20020173634A1 (en) 2000-10-31 2002-11-21 Fraser Malcolm J. Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
US20100221824A1 (en) 2000-10-31 2010-09-02 University Of Notre Dame Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggyBac
US20040077572A1 (en) 2002-04-22 2004-04-22 Regents Of The University Of Minnesota Transposon system and methods of use
US20050003542A1 (en) 2003-06-04 2005-01-06 Kay Mark A. Enhanced sleeping beauty transposon system and methods for using the same
US20060026699A1 (en) 2004-06-04 2006-02-02 Largaespada David A Methods and compositions for identification of genomic sequences
US20070204356A1 (en) 2006-02-28 2007-08-30 University Of Notre Dame Du Lac PiggyBac constructs in vertebrates
US20110117072A1 (en) 2007-07-04 2011-05-19 Max-Delbruck-Centrum Fur Molekulare Medizin Hyperactive variants of the transposase protein of the transposon system sleeping beauty

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