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WO2000026396A1 - Methode de recombinaison et agents utiles dans ladite methode - Google Patents

Methode de recombinaison et agents utiles dans ladite methode Download PDF

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WO2000026396A1
WO2000026396A1 PCT/AU1999/000835 AU9900835W WO0026396A1 WO 2000026396 A1 WO2000026396 A1 WO 2000026396A1 AU 9900835 W AU9900835 W AU 9900835W WO 0026396 A1 WO0026396 A1 WO 0026396A1
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recombination
recbcd
nucleotide sequences
nucleotide sequence
host cell
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Panos Ioannou
Kumaran Narayanan
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MURDOCH INSTITUTE
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MURDOCH INSTITUTE
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Priority to EP99950404A priority Critical patent/EP1127152A4/fr
Priority to AU63210/99A priority patent/AU6321099A/en
Priority to JP2000579768A priority patent/JP2002528129A/ja
Publication of WO2000026396A1 publication Critical patent/WO2000026396A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

  • the present invention relates generally to a method for the recombination of at least two nucleic acid sequences and agents useful for same. More particularly, the present invention contemplates a method of recombining, in a host cell which expresses the recBCD nuclease or a functional derivative thereof, a circular nucleic acid sequence with a linear nucleic acid sequence.
  • the method of the present invention is useful, inter alia, for the modification of bacterial artificial chromosomes by homologous recombination with a linear DNA sequence.
  • Genomic sequences are oftern much longer than coding sequences. Although the functions of most non-coding sequences are not known, it is clear that may play a crucial role in regulation of gene expression, stability and evolution. In contrast to cDNA constructs driven by unrelated or viral promoters, gene transfer of large genomic fragments shows correct temporal- and tissue-specific gene expression. 1 7
  • Yeast artifical chromosomes have provided genomic clones of many disease genes. 8"10 Transgenic models with YACs for a number of human genetic loci have demonstrated the usefulness of such large genomic fragments to produce accurate models for various diseases. n'14 The high efficiency of homologous recombination in yeast has been of crucial significance in the genetic manipulation of YACs for functional analysis, 3,8,15 the circularisation of YACs into PACs or BACs 16,!7 and the generation of larger YACs from smaller overlapping YACs. 18 There are, however, several limitations with the YAC system which have seriously impeded its use. YACs have a high degree of clonal instability and chimerism.
  • yeast homologous recombination system for introducing modifications in YACs or for TAR cloning
  • 22 is the difficulty in regulating the extent of homologous recombination and the need to screen large numbers of "recombinant" clones in order to identify the correct products.
  • BACs/PACs are used increasingly for long-range physical mapping, 25,26 positional cloning of disease genes, 27 whole genome sequencing projects 28"30 and functional studies.
  • 3I 32 High quality BAC/PAC genomic libraries are much easier to construct than YAC libraries because of greater cloning efficiency in bacteria.
  • BACs/PACs are maintained at 1-2 copies per cell in a well-defined recombination-deficient E. coli strain, DH10B, where they exhibit high clonal stability over many generations. Although they carry inserts up to about 300kb in size, they can be purified in large quantities for functional studies through conventional bacterial plasmid isolation methods. The genomic inserts in these clones are large enough to preserve the integrity of most human genetic loci and are thus ideal for functional studies.
  • RecA-assisted restriction endonuclease (RARE) cleavage approach has also been used successfully to introduce modifications in a number of PI and BAC clones, 35 although it is technically a very restrictive procedure. Since both the PAC and BAC cloning systems include a loxP site on the backbone of each vector, a number of workers have targeted the insertion of eukaryotic selectable markers and reporter genes into this site with ere recombinase. 36,37
  • Yang et al 38 reported targeted modification of a 131kb BAC using the wild type recA gene in a shuttle plasmid with a temperature-sensitive origin of replication, as a means of conferring transient recombination proficiency to DH10B cells. There was evidence, however, for significant rearrangements of clones using this procedure, necessitating characterisation of recombinant clones by Southern blot to identify the correct products.
  • An alternative approach using the CBTS strain of E. coli, which carries a temperature- sensitive amber mutation in the recA gene, has also been used to introduce modifications in a 230kb BAC containing the intact mouse CMV genome.
  • the inventors sought to develop a method for the controlled homologous recombination, in vivo, of two nucleic acid sequences such as a circular DNA sequence with a linear DNA sequence.
  • the inventors have modified BACs by expressing in a host cell, a modulatable recE/recT recombination system together with modulatable levels of a recBCD nuclease inhibitor thereby permitting the homologous recombination of a DNA sequence with the BAC.
  • Expression of the recBCD nuclease inhibitor acts to inhibit the functional activity of endogenously produced recBCD nuclease.
  • the capacity to modulate and coordinate expression of both the recE/recT homologous recombination system and the recBCD nuclease inhibitor facilitates the control of homologous recombination events and minimises random and unwanted recombination events.
  • the capacity to modulate and coordinate expression of the homologous recombination system and the recBCD nuclease inhibitor facilitates the, in vivo, induction of homologous recombination events both where at least one of the nucleic acid sequences which is the subject of recombination is located in an F-plasmid, such as a BAC, or where the host cell is sensitive to the over expression of recombination system molecules, or recBCD inhibitors such as gam.
  • the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • Another aspect of the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD, recE, recT and a nucleotide sequence encoding a recBCD inhibitor or functional derivatives thereof, the expression of which recE, recT and nucleotide sequence encoding a recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • Yet another aspect of the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD, recE, recT and gam or functional derivatives thereof, the expression of which recE, recT and gam is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • Still another aspect of the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD, recE, recT and gam or functional derivatives thereof, the expression of which recE, reel and gam is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • Still yet another aspect of the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • a further aspect of the present invention provides a method for facilitating homologous recombination between a circular nucleotide sequence and a linear nucleotide sequence said method comprising inducing recombination between said circular nucleotide sequence and said linear nucleotide sequence in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the circular nucleotide sequence with the linear nucleotide sequence.
  • Another further aspect of the present invention provides a method for facilitating homologous recombination between a circular nucleotide sequence and a linear nucleotide sequence said method comprising inducing recombination between said circular nucleotide sequence and said linear nucleotide sequence in a DH10B cell or homolog or mutant thereof which is capable of expression recBCD, recE, recT and gam or functional derivatives thereof, the expression of which recE, recT and gam is modulatable, by culturing the DH10B cell or homolog or mutant thereof under conditions sufficient to facilitate the homologous recombination of the circular nucleotide sequence with the linear nucleotide sequence.
  • Still another further aspect of the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences wherein at least one of said nucleotide sequences comprises an F-plasmid portion said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivative thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modultable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • Still yet another aspect of the present invention provides a method for facilitating homologous recombination between a BAC and a linear nucleotide sequence said method comprising inducing recombination between said BAC and said linear nucleotide sequence in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the BAC with the linear nucleotide sequence.
  • Another aspect of the present invention contemplates a method for facilitating the homologous recombination of at least two nucleotide sequences in a host cell said method comprising the steps of:
  • Still another aspect of the present invention contemplates a method of producing a microorganism useful for facilitating homologous recombination between at least two nucleotide sequences said method comprising genetically manipulating a host cell such that it is capable of expressing recBCD, modulatable levels of an exonuclease, a recombination protein and a gene encoding a recBCD inhibitor and said at least two other nucleotide sequences.
  • Still another further aspect of the present invention contemplates a cell capable of facilitating homologous recombination between at least two nucleotide sequences said cell comprising nucleotide sequences encoding an exonuclease, a recombination protein and a gene encoding a recBCD inhibitor, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, recBCD and said at least two nucleotide sequences.
  • the present invention contemplates nucleic acid molecules homologously recombined by the method of the present invention.
  • a further aspect of the present invention contemplates a pharmaceutical composition comprising modified nucleic acid molecules generated by the method of the present invention together with one or more pharmaceutically acceptable carriers and/or diluents.
  • kits for facilitating, in a host cell, the homologous recombination of at least two nucleotide sequences said kit comprising compartments adapted to contain any one or more of nucleotide sequences encoding an exonuclease, a recombination protein, a recBCD inhibitor or functional derivatives thereof, and reagents useful for facilitating homologous recombination. Further compartments may also be included, for example to receive biological samples such as any one or more of the nucleotide sequences which are to be recombined, the host cells or host cells already stably transformed with one or more of the nucleotide sequences which are to be recombined.
  • Still yet another further aspect of the present invention is directed to a kit for facilitating, in a host cell, the homologous recombination of at least two nucleotide sequences
  • said kit comprising compartments adapted to contain any one or more of an exonuclease, a recombination protein, a recBCD inhibitor, or functional derivative thereof and reagents useful for facilitating homologous recombination.
  • Further compartments may also be included, for example to receive biological samples such as any one or more of the nucleotide sequences which are to recombined, the host cell or host cells already stably transformed with one or more of the nucleotide sequences which are to recombined.
  • Figure 1 is a schematic representation of the pEBAC140 vector map, showing its main features.
  • the vector is based on the backbone of the F-plasmid pBeloBACl 1 , 23
  • the hygromycin and thymidine kinase genes provide for selection in eukaryotic cells, while the oriP and EBNA-1 genes from Epstein-Barr virus facilitate episomal maintenance.
  • the vector contains a unique rare cutter multicloning site inserted in frame into the lacZ gene to allow for blue/white selection of recombinants and for recovery of genomic inserts by any of a number of different cutters which flank the Bam ⁇ l cloning sites.
  • a modified pUC19-Xho is inserted into the Xhol site in later versions of the vector to render it multicopy and to facilitate cloning applications.
  • Figure 2 is a schematic representation of the pEBAC/148 which was generated by insertion of a 185kb genomic fragment from a PAC clone carrying the intact ⁇ -globin genomic region into the N ⁇ tl sites of the pEBAC140 vector. The position of targeting the integration of the kanamycin resistance gene in this clone through homologous recombination and in the original pEBAC140 vector is indicated.
  • FIG. 3 is a schematic representation of a map showing the main features of the pGETrec plasmid.
  • a PCR product containing the gam gene of bacteriophage ⁇ was inserted into the pBAD24-recET plasmid 45 to produce pGETrec.
  • the gam gene is thus under the control of the arabinose promoter, together with the recE and recT genes.
  • FIG 4 is a schematic representation of the oligonucleotide primers used.
  • PCRkan50 (1262bp) was generated by amplification of kanamycin gene from pCYPAC2 with primers kan50F and kan50R.
  • Primers kan50F and kan50R also carry 50-mer targeting regions homologous to the sequences flanking the unique 5 ⁇ 11071 site on the vector target sequence.
  • PCRkanl40 was generated with primers KF and KR after homologous recombination of PCRkan50 into the pEBAC140 vector.
  • the expected size of the PCR products with primers KF/KR is 288bp and 1450bp in the absence and presence of the kanamycin gene respectively.
  • the expected size of the corresponding PCR products with primers KA/KB is 393bp and 1555bp.
  • FIG. 5 is a schematic representation of the "GET recombination" method for integration of PCR fragments through homologous recombination into BACs/PACs in DH10B cells.
  • PCR primers are each designed to carry a 50-mer homology region to the targeted sites in a PAC or BAC, together with specific sequences at their 3' -ends for amplifying a DNA fragment with a selectable marker. Additional sequences or markers may also be included.
  • the PCR product generated by these primers is electroporated into electrocompetent DH10B cells, carrying both the BAC or PAC clone of interest and the pGETrec plasmid, which are transiently induced with 0.2% L-arabinose during preparation.
  • the modified BAC or PAC clone is purified away from pGETrec by miniprep DNA isolation and electroporation into DH10B cells or by streaking in media with an antibiotic.
  • Figure 6 is a photographic representation of PCR analysis of 20 independent Cm r Km r colonies carrying pEBAC/148: :kanl40. One microlitre of an overnight culture grown from each Cm r Km r colony was added into the PCR reaction with PCR primers KF and KR. The PCR products were resolved using a 1.5%(w/v) agarose gel. Lanes: 1 and 24, 1-kb DNA ladder standards; lanes 2-21, 20 independent CmT m" colonies; lane 22, parent pEBAC/148 clone; lane 23, negative control (no cells).
  • Figure 7 is a photographic representation of PFGE analysis of 4 randomly picked C ⁇ fK ⁇ colonies (A-D) after integration of PCRkanl40 into pEBAC/148 by homologous recombination. DNA from each clone was re-electroporated into DH10B cells. Duplicate secondary colonies were used for miniprep extraction of DNA and restriction enzyme digestion with Notl or Xhol.
  • Notl-digestion M, low range PFG marker; Lanes 1-8, duplicate pEBAC/148: :kanl40 clones; P, parent pEBAC/148 clone.
  • PFGE conditions 6V/cm, switch time of 0.1-25.0s, 14°C, for 17 hours.
  • X/r ⁇ l-digestion Ml, low range PFG marker; Lanes 1-8, duplicate pEBAC/148::kanl40 clones; P, parent pEBAC/ 148 clone; M2, midrange I PFG marker.
  • PFGE conditions 6V/cm, switch time of 0.1-8.0s, 14°C, for 18 hours.
  • the present invention is predicated, in part, on the development of a method for the homologous recombination of at least two nucleotide sequences in a host cell by introducing into a host cell a nucleotide sequence which expresses modulatable levels of an exonuclease, a recombination protein and a recBCD nuclease inhibitor.
  • Expression of a recBCD nuclease inhibitor is necessary to inhibit the linear DNA degradation activity of the recBCD nuclease which is endogenously expressed by host cells such as the E. coli strain DH10B.
  • the method of the present invention is therefore based on the modulatable and coordinate induction of a homologous recombination system and the inhibition of recBCD nuclease activity.
  • the advent of a modulatable and coordinate induction system has particularly facilitated the application of homologous recombination methodology in host cells which are sensitive to the over-expression of either one or more recombination system molecules or recBCD inhibitors. It is also particularly applicable in situations where the homologous recombination of a nucleic acid sequence comprising a F-plasmid portion is desired.
  • the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • exonuclease should be understood as a reference to a molecule which facilitates homologous recombination by exposing single stranded regions on a double stranded nucleic acid sequence which regions are necessary to effect recombination between this region and a complementary region of another nucleic acid sequence.
  • the exonuclease is one which is suitable for use with the selected recombination protein.
  • exonucleases suitable for use in the present invention include, but are not limited, the Red ⁇ recombination system exonuclease termed exo, and the recE/recT recombination system molecule exonuclease termed recE, or functional derivatives thereof.
  • exonuclease is recE or functional derivative thereof.
  • Reference to a "recombination protein" should be understood as a reference to a peptide, polypeptide or protein which facilitates homologous recombination by mediating the recombination between the single stranded region of one nucleic acid sequence (such as the single stranded region generated by the exonuclease) and the complementary region of another nucleic acid sequence.
  • the recombination protein include, but are not limited to, the Red ⁇ recombination protein bet and the recE/recT recombination protein recT, or functional derivatives thereof.
  • the nucleotide sequence encoding recE and recT are herein referred to as recE and recT, respectively.
  • the present invention more particularly provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD, recE, recT and a nucleotide sequence encoding a recBCD inhibitor or functional derivatives thereof, the expression of which recE, recT and nucleotide sequence encoding a recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • expression refers to the transcription and translation of a nucleotide sequence resulting in the synthesis of a peptide, polypeptide or protein.
  • references to "a gene encoding a recBCD inhibitor” should be understood as a reference to a nucleotide sequence which encodes an expression product which can either inhibit or sufficiently down-regulate the activity of the recBCD nuclease such that linear DNA sequences are not degraded by this nuclease and their homologous recombination is thereby able to occur.
  • recBCD inhibitors suitable for use in the present invention include, but are not limited to, gam, P22Abc protein or SSB protein or functional derivatives thereof .
  • Preferably said recBCD inhibitor is gam.
  • the nucleotide sequence encoding gam is herein referred to as gam.
  • the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD, recE, recT and gam or functional derivatives thereof, the expression of which recE, recT and gam is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • host cell should be understood as a reference to any prokaryotic or eukaryotic cell which can be transformed or transfected with a nucleotide sequence.
  • host cells suitable for cloning and/or expression of nucleotide sequences such as host cells which are used to create gDNA or cDNA libraries or those which are used for cloning a vector which comprises a DNA sequence insert of interest.
  • the host cell is preferably one which expresses the recBCD nuclease (also termed "exonuclease V”) which, inter alia, degrades linear, double stranded DNA.
  • the host cell may be expressing recBCD either due to the expression of a naturally occurring recBCD gene or due to the transformation or transfection of the host cell with a nucleic acid molecule which encodes recBCD.
  • strains of host cell are sensitive to over-production, such as constitutive production, of a recBCD inhibitor and/or one or more molecules of a homologous recombination system.
  • sensitive is meant that the host cell is compromised in some way such as, for example, reduction in its viability or the undesirable modulation of one or more of its functional activities.
  • constitutive production of the recBCD inhibitor, gam in DH10B host cells is found to be toxic in that the viability of DH10B cells is reduced.
  • the host cell is a host cell which is sensitive to the constitutive production of one or more of a recBCD inhibitor, an exonuclease or a recombination protein. Even more preferably said host cell is DH10B or mutant or homolog thereof.
  • the present invention extends to the use of mutants and homologs of host cells.
  • the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • said exonuclease is recE
  • said recombination protein is recT
  • said recBCD inhibitor is gam.
  • nucleotide sequences should be understood as a reference to any two or more nucleotide sequences which are to be homologously recombined by the method of the present invention.
  • Those nucleotide sequences may be separate nucleic acid molecules such as, for example, two circular vectors (such as two plasmids), a plasmid and a linear DNA sequence or a chromosome and a linear DNA sequence.
  • the nucleotide sequences may be two portions of a single nucleic acid molecule such as, for example, the linear and circular nucleotide sequence portions of the single nucleotide molecule generated during rolling circle replication.
  • the nucleotide sequences are a circular nucleotide sequence and a linear nucleotide sequence.
  • a "circular" nucleotide sequence should be understood as a reference to the circular nucleotide sequence portion of any nucleotide molecule.
  • the circular nucleotide sequence may be completely circular, such as a plasmid, or it may be partly circular, such as the circular portion of the nucleotide molecule generated during rolling circle replication.
  • the "circular" nucleotide sequence corresponds to the circular portion of this molecule.
  • the circular nucleic acid sequence is a artificial chromosome of the type BAC or PAC.
  • linear nucleotide sequence should be understood as a reference to any nucleotide sequence which is in essentially linear form.
  • the linear sequence may be a linear nucleotide molecule or it may be a linear portion of a nucleotide molecule which also comprises a non-linear portion such as a circular portion.
  • linear nucleotide sequences include, but are not limited to, PCR products, excision products, synthesized DNA or the linear portion of a nucleotide molecule generated during rolling circle replication. Recombination of the linear nucleotide sequence with the circular nucleotide sequence may, for example, introduce into the circular nucleotide sequence a selectable marker or a specific mutation.
  • homologous recombination of the present invention may occur between nucleotide sequences which are introduced into a cell or it may occur between nucleotide sequences which are naturally found in the cell and one or more introduced nucleotide sequences.
  • the present invention preferably provides a method for facilitating homologous recombination between a circular nucleotide sequence and a linear nucleotide sequence said method comprising inducing recombination between said circular nucleotide sequence and said linear nucleotide sequence in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the circular nucleotide sequence with the linear nucleotide sequence.
  • said host cell is a DH10B cell.
  • said exonuclease is recE
  • said recombination protein is recT
  • said recBCD inhibitor is gam or functional derivatives thereof.
  • the present invention provides a method for facilitating homologous recombination between a circular nucleotide sequence and a linear nucleotide sequence said method comprising inducing recombination between said circular nucleotide sequence and said linear nucleotide sequence in a DH10B cell or homolog or mutant thereof which is capable of expression recBCD, recE, recT and gam or functional derivatives thereof, the expression of which recE, recT and gam is modulatable, by culturing the DH10B cell or homolog or mutant thereof under conditions sufficient to facilitate the homologous recombination of the circular nucleotide sequence with the linear nucleotide sequence.
  • nucleic acid sequences of the present invention are preferably derivable from the human genome but genomes and nucleotide sequences from non-human animals and plants are also encompassed by the present invention.
  • Non-human animals contemplated by the present invention include primates, livestock animals (eg. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (eg. mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals (eg. dogs, cats), birds (eg. chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals (eg. foxes, kanagaroos, dingoes).
  • livestock animals eg. sheep, cows, pigs, goats, horses, donkeys
  • laboratory test animals eg. mice, rats, guinea pigs, hamsters, rabbits
  • domestic companion animals eg. dogs
  • the homologous recombination system of the present invention is suitable for recombining at least two nucleotide sequences where at least one of said nucleotide sequences comprises an F-plasmid portion.
  • Reference to a nucleotide sequence which "comprises" an F-plasmid portion should be understood as a reference to a nucleotide sequence which is fused, bound or otherwise associated with all or part of an F-plasmid.
  • the nucleotide sequence of interest may be located within a vector, the backbone of which comprises sequence corresponding to all or part of the F-plasmid.
  • the F-plasmid based BAC for example comprises genomic sequences of interest which are located in a vector expressing the wild-type F-plasmid origin of replication and the par A, par B and par C encoding nucleotide sequences.
  • the present invention provides a method for facilitating homologous recombination between at least two nucleotide sequences wherein at least one of said nucleotide sequences comprises an F-plasmid portion said method comprising inducing recombination between said at least two nucleotide sequences in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivative thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modultable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the at least two nucleotide sequences.
  • said nucleotide sequence which comprises an F-plasmid portion is a BAC.
  • said host cell is DH10B or mutant or homolog thereof.
  • BAC/PAC cloning system has facilitated the study of the mammalian genome via the gene transfer of large genomic fragments which show correct temporal and tissue-specific gene expression.
  • Large-insert BAC/PAC cloning systems have therefore been used extensively for long-range physical mapping, positional cloning of disease genes, whole genome sequencing projects and functional studies.
  • BAC/PAC E. coli libraries have been created for human genomic DNA as well as for the genomic DNA of other animal and plant species including baboon, canine, bovine, ovine, goat and rice.
  • BACs/PACs are generally maintained at 1-2 copies per cell in the well defined recombination-deficient E.coli strain DH10B.
  • a particularly preferred embodiment of the present invention is directed to the modification of a BAC or PAC via the homologous recombination of a linear DNA segment into the BAC or PAC.
  • the present invention provides a method for facilitating homologous recombination between a BAC and a linear nucleotide sequence said method comprising inducing recombination between said BAC and said linear nucleotide sequence in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the BAC with the linear nucleotide sequence.
  • said host cell is a DH10B cell or mutant or homolog thereof.
  • said exonuclease is recE
  • said recombination protein is recT
  • said recBCD inhibitor is gam.
  • the present invention provides a method for facilitating homologous recombination between a PAC and a linear nucleotide sequence said method comprising inducing recombination between said PAC and said linear nucleotide sequence in a host cell which is capable of expressing recBCD and nucleotide sequences encoding an exonuclease, a recombination protein and a recBCD inhibitor or derivatives thereof, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, by culturing the host cell under conditions sufficient to facilitate the homologous recombination of the PAC with the linear nucleotide sequence.
  • said host cell is a DH10B cell or mutant or homolog thereof.
  • said exonuclease is recE
  • said recombination protein is recT
  • said recBCD inhibitor is gam.
  • the method of the present invention may involve the transformation of the subject host cell with any one or more of recBCD, nucleotide sequences encoding an exonuclease and a recombination protein, a nucleotide sequence encoding a recBCD inhibitor and the at least two nucleotide sequences. How many of these molecules will require introduction into the host cell to facilitate the operation of the method of the present invention will depend on the particular application in issue. For example, with respect to the exemplified recE/recT recombination system it may be desirable to use a population of host cells from a commercially available BAC or PAC DH10B library.
  • the BAC or PAC molecule may be modified by recombining it with a linear nucleotide sequence which is introduced into the commercially available DH10B clones from the library following tranformation with a nucleic acid molecule encoding recE, recT and gam. DH10B cells endogenously express recBCD.
  • a nucleic acid molecule encoding recE, recT and gam. DH10B cells endogenously express recBCD.
  • the use of host cells which already express recBCD, recE, recT and a nucleotide sequence encoding a recBCD inhibitor will only require transformation with the nucleotide sequences which are to be homologously recombined and which are not already present in the host cell.
  • These host cells may be expressing recBCD, recE, recT and a nucleotide sequence encoding a recBCD inhibitor either due to the cell having been engineered to express any one or more of these molecules or due to the cell endogenously expressing these molecules.
  • the expression of the exonuclease, the recombination protein and the recBCD inhibitor is modulatable.
  • modulatable it is meant that the expression of the genes encoding these proteins can be up-regulated or down-regulated at the transcriptional or translation level. This modulation can be achieved by any one of a number of techniques including, but not limited to, regulating promoter expression or regulating methylation.
  • DH10B cells transformed with the BAC nuleic acid molecule, pEBAC140 are transfected with the inducible expression plasmid pGETrec which comprises the nucleic acid molecules encoding recE, recT and gam under the control of the inducible L-arabinose promoter.
  • pGETrec which comprises the nucleic acid molecules encoding recE, recT and gam under the control of the inducible L-arabinose promoter.
  • the present invention is exemplified by the introduction, in a host cell, of a single plasmid which comprises the nucleic acid sequences recE, recT and gam, under the control of an inducible promoter (i.e. the nucleic acid molecules encoding recE, recT and gam are operably linked to a common promoter), the method of the present invention should be understood to extend to the use of two or more vectors separately comprising recE, recT and gam.
  • an inducible promoter i.e. the nucleic acid molecules encoding recE, recT and gam are operably linked to a common promoter
  • recE, recT and gam it would be necessary to ensure that the induction of expression of recE, recT and gam could be achieved simultaneously, for example, by utilising the same inducible promoter with each of recE, reel and gam.
  • the recE/recT homologous recombination system would therefore become functionally active simultaneously with the gam inhibition of recBCD nuclease activity.
  • references to "facilitating" homologous recombination should be understood as a reference to inducing, enhancing or otherwise contributing to the functional operation of the homologous recombination system.
  • the recE and recT expression products induce recE/recT based recombination while the gam expression product inhibits the activity of the recBCD nuclease, which degrades linear DNA thereby preventing its recombination.
  • pGETrec can co-exist stably with BAC/PAC in E. coli DH10B cells.
  • homologous recombination at high efficiency can be achieved between linear nucleic acid fragments and target circular nucleic acid molecules. Efficiency of homologous recombination is improved where the length of homology is increased from 50bp to 140bp. Further, it is thought that significant increases in efficiency of homologous recombination with BAC/PAC can be achieved by use of non-adjacent sequences in the targetted homology region.
  • the present invention should be understood to extend to the induction of in vivo homologous recombination by introducing into the host cell the proteins or functional derivatives thereof which facilitate homologous recombination rather than introducing into the host cell nucleic acid molecules which are induced to express these proteins.
  • the coordinated introduction of recE, recT and gam proteins, or functional derivatives thereof, into the host cell together with the nucleotide sequences which are to be the subject of recombination is envisaged.
  • methods for performing the present invention wherein some of the homologous recombination system proteins are expressed in the host cell while the remainder are introduced into the cell in protein form.
  • expression within the host cell of recE and recT or functional derivatives thereof together with the coordinated administration of gam protein is envisaged.
  • This aspect of the present invention should be understood to extend to the delivery of any one or more of the exonuclease, recombination protein or recBCD inhibitor or functional derivatives thereof.
  • Derivatives include fragments, parts, portions, chemical equivalents, mutants, homologues, analogues, mimetics from natural, synthetic or recombinant sources including fusion proteins. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins.
  • the derivatives of said nucleotide sequences and expression products include fragments having particular epitopes or parts of the entire component fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • said components or derivative thereof may be fused to a molecule to facilitate its entry into a cell.
  • Analogs of said components contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogs.
  • nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
  • the derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; per formic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethy lpy rocarbonate .
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid contemplated herein is shown in Table 1.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the method of the present invention is useful as a simple and efficient technique for allowing the introduction of any desirable modification at any position in a circular DNA clone such as a BAC or PAC, without unwanted re-arrangements. Modifications such as the introduction of selectable markers or specific mutations corresponding to those found to cause human pathology are examples of the use to which this method can be put.
  • the method of the present invention reduces the need for transferring cloned circular DNA molecules such as BACs/PACs to other E. coli strains for homologous recombination. It is also reduces the need for the creation of special shuttling vectors.
  • the procedure is simple enough that it allows repeated use on any single clone to form complex changes.
  • the procedure can be modified to allow the inclusion of a reporter gene or any other desired sequence into circular nucleic acid molecules.
  • these approaches allow for very sensitive assay systems for monitoring the impact of various sequence elements and mutations on the tissue and developmental specificity of gene transcription, splicing and expression from intact functional loci.
  • These techniques facilitate the development of pharmacological approaches aimed at altering the expression of genes with increased tissue and locus specificity, as well as human artificial chromosomes, as a means of alleviating or curing a variety of human genetic diseases.
  • the creation of accurate animal models with normal and mutant functional loci also facilitates gene therapy approaches involving gene supplementation or correction of the mutant locus.
  • Another aspect of the present invention contemplates a method for facilitating the homologous recombination of at least two nucleotide sequences in a host cell said method comprising the steps of:
  • said exonuclease is recE
  • said recombination protein is recT
  • said gene encoding a recBCD inhibitor is gam.
  • said at least two nucleotide sequences are a nucleotide sequence comprising an F-plasmid portion and a linear nucleotide sequence.
  • said nucleotide sequence comprising an F-plasmid portion is BAC.
  • said host cell is DH10B or mutant or homolog thereof.
  • Still another aspect of the present invention contemplates a method of producing a microorganism useful for facilitating homologous recombination between at least two nucleotide sequences said method comprising genetically manipulating a host cell such that it is capable of expressing recBCD, modulatable levels of an exonuclease, a recombination protein and a gene encoding a recBCD inhibitor and said at least two other nucleotide sequences.
  • said exonuclease is recE
  • said recombination protein is recT
  • said gene encoding a recBCD inhibitor is gam.
  • said at least two nucleotide sequences are a nucleotide sequence comprising an F-plasmid portion and a linear nucleotide sequence. Still more preferably, said circular nucleotide sequence comprising an F-plasmid portion is BAC.
  • said host cell is DH10B or mutant or homolog thereof.
  • Still another aspect of the present invention contemplates a cell capable of facilitating homologous recombination between at least two nucleotide sequences said cell comprising nucleotide sequences encoding an exonuclease, a recombination protein and a gene encoding a recBCD inhibitor, the expression of which exonuclease, recombination protein and recBCD inhibitor is modulatable, recBCD and said at least two nucleotide sequences.
  • said exonuclease is recE
  • said recombination protein is recT
  • said gene encoding a recBCD inhibitor is gam.
  • said at least two nucleotide sequences are a nucleotide sequence comprising an F-plasmid portion and a linear nucleotide sequence.
  • said nucleotide sequence comprising an F-plasmid portion is BAC.
  • said cell is DH10B or mutant or homolog thereof.
  • the present invention contemplates nucleic acid molecules homologously recombined by the method of the present invention.
  • modified nucleic acid molecules are modified BACs or modified PACs.
  • the present invention also extends to the use of said modified nucleic acid molecules in the treatment and/or diagnosis of patients.
  • Methods of treatment include gene therapy regimens.
  • the present invention also extends to methods of screening which utilise said modified nucleic acid molecules.
  • another aspect of the present invention contemplates a pharmaceutical composition
  • a pharmaceutical composition comprising modified nucleic acid molecules generated by the method of the present invention together with one or more pharmaceutically acceptable carriers and/or diluents.
  • an expression vector comprising a genetic construct substantially as set forth in Figure 3 or functional derivative thereof.
  • kits for facilitating, in a host cell, the homologous recombination of at least two nucleotide sequences said kit comprising compartments adapted to contain any one or more of nucleotide sequences encoding an exonuclease, a recombination protein, a recBCD inhibitor or functional derivatives thereof, and reagents useful for facilitating homologous recombination. Further compartments may also be included, for example to receive biological samples such as any one or more of the nucleotide sequences which are to be recombined, the host cells or host cells already stably transformed with one or more of the nucleotide sequences which are to be recombined.
  • Still yet another aspect of the present invention is directed to a kit for facilitating, in a host cell, the homologous recombination of at least two nucleotide sequences
  • said kit comprising compartments adapted to contain any one or more of an exonuclease, a recombination protein, a recBCD inhibitor, or functional derivative thereof and reagents useful for facilitating homologous recombination.
  • Further compartments may also be included, for example to receive biological samples such as any one or more of the nucleotide sequences which are to recombined, the host cell or host cells already stably transformed with one or more of the nucleotide sequences which are to recombined.
  • kits according to this aspect of the present invention should also be understood to extend to kits which comprise a combination of nucleotide sequences encoding one or more recombination system proteins or the recombination system proteins themselves.
  • the recE pathway of homologous recombination between linear and circular substrates is active in the sbcA recBC strains JC8679 and JC9604. 42"45 This pathway is recA independent, 46 since the recT gene appears to fulfil the role of recA in pairing of homologous sequences. 47 Precise homologous recombination of PCR fragments of a kanamycin resistance (Km*) gene (PCRkan50) carrying 50bp flanking homology arms, into the backbone of the chloramphenicol resistant (Cm 1 ) pEBAC140 vector in these cells was obtained, to generate pEBAC140::kan50.
  • Km* kanamycin resistance
  • Cm 1 chloramphenicol resistant
  • the pBAD24-recET plasmid which carries the recE and recT genes under the control of an inducible arabinose promoter, was electroporated into DH10B(pEBAC140) cells and clones carrying both plasmids were selected on ampicillin and chloramphenicol media.
  • a PCR fragment carrying the gam gene was inserted into the pB AD24-recET plasmid, to produce plasmid pGETrec (Fig. 3).
  • plasmid pGETrec Fig. 3
  • the inducible expression plasmid, pGETrec was introduced into electrocompetent DH10B cells already carrying pEBAC140.
  • DNA was isolated from six of these CmT ⁇ colonies. A large and variable amount of the multicopy pGETrec plasmid was recovered together with the single copy pEBAC/H8::kanH0 BAC and interfered with interpretation of restriction digests of the BAC. Re-electroporation of 1 ⁇ l of DNA from four clones into DH10B cells resulted in many independent secondary CmT nf colonies from each clone. DNA was isolated from two independent secondary clones of each primary clone and analysed by pulsed field gel electrophoresis after digestion with Notl or Xhol (Fig. 7).
  • the 70kb fragment Since there is no Xhol site on the backbone of the parent vector, the 70kb fragment must be derived from the backbone of the vector and adjoining globin sequences. After integration of PCRkanHO into the vector backbone by homologous recombination, the 70kb fragment is cut into two smaller fragments at the single Xhol site which is present in the kanamycin gene.
  • PCRkan50 into DH10B(pGETrec, pEBAC/148) cells which were only induced for 40 min with L-arabinose during preparation. More than 100 Cm r Km r colonies were obtained from each electroporation (Table 1, Experiments 5, 6). Direct PCR screening of 20 independent overnight cultures from these Cm r Km r colonies, using primers HOKF and HOKR, generated the correct 1450bp product.
  • the second generation pEBACHO BAC/PAC cloning vector (Fig. 1) combines a number of features from the first generation PAC 24 and BAC 23 cloning systems, as well as the oriP and EBNA-I of Epstein Barr virus from the HAEC system. 48 It also contains a number of additional features, including a rare cutter multicloning region.
  • pEBAC/148 (Fig. 2) was prepared from pEBACHO by retrofitting a 185kb genomic fragment carrying the entire ⁇ - globin locus, using procedures based on the PAC cloning protocols. 49 Briefly, the RPCI 1 human total genomic PAC library 49 was screened by PCR primers from the 5'- and 3'-ends of the ⁇ -globin locus. A PAC clone carrying a 185kb genomic insert was identified
  • the 185kb genomic insert was isolated as a single fragment by Notl digestion, pulsed field gel electrophoresis (CHEF-DRII, BIO- RAD Labs., Hercules, CA) and gelase (Epicentre Technologies, Madison, WI) digestion.
  • the pEBACHO vector was also digested by Notl, dephosphorylated and gel purified before ligation to the purified 185kb globin fragment.
  • Three independent pEB AC/148 clones were isolated after electroporation into DHl OB cells.
  • the gam gene of bacteriophage ⁇ was PCR amplified using primers gam-F, 5'-
  • AGGTAGGATCCACCATGGATATTAATACTGAAAC-3' (SEQ ID ⁇ O:l), and gam-R, 5'-ACTGAGGATCCTCGTTTTATACCTCTGAATCAATAT-3' (SEQ ID NO:2), from plasmid pTP223, 41 digested with BamHl, and cloned into the BgHl site of pBAD24-recET. 43 A clone with the correct orientation of the gam gene relative to the arabinose promoter was designated pGETrec (Fig. 3).
  • DHl OB cells (GIBCO-BRL, Gaithersburg, MD) carrying both pGETrec and either pEBACHO or pEBAC/148 were diluted 50-fold into 250ml fresh LB medium containing lOO ⁇ g/ml ampicillin and 12.5 ⁇ g/ml chloramphenicol.
  • L-arabinose (SIGMA, St. Louis, MO) was added to a final concentration of 0.2% (w/v) when the OD600 of the cells reached 0.40 - 0.42.
  • Electrocompetent cells were prepared by harvesting at OD 600 0.55 to 0.60 using the protocol recommended by the manufacturer. The final cell pellet was resuspended in a total volume of 400 to 500 ⁇ l of 10% glycerol. Cells were aliquoted to pre-chilled 1.5ml microfuge tubes (40 ⁇ l/tube) and frozen in a dry ice- ethanol bath. The frozen cells were stored at -70°C.
  • kanamycin gene from vector pCYPAC2 49 was amplified with the following primers: kan50F 5'-ACCACATACGTTCCGCCATTCCTATGCGATGCACATGCTGTATGCCGGTACA AGAAATCACAGCCGAAGC-3' (SEQ ID NO:3) and kan50R
  • each primer sequence corresponds to the sequence in the upper and lower strands of the pEBACHO vector bordering the unique BstX 1071 site, while the last 20 nucleotides of each primer correspond to sequences flanking the kanamycin gene in the pCYPAC2 vector.
  • PCRkan50 The PCR product, PCRkan50, is 1262bp long and carries the kanamycin gene and promoter region, as well as 50mer sequences at each end for targeting its integration through homologous recombination into the Bst ⁇ 1071 site on the pEBACHO vector (Fig. 4). This was used to generate pEBACH0::kan50 by targeting the integration of PCRkan50 into pEBACHO in E. coli JC8679 cells.
  • PCRkanHO is 1450bp long and was generated by PCR from pEBAC140::kan50 by using primers 140KF 5'-ATCTGGGAAGTGACGGACAG-3' (SEQ ID NO:5) and 140KR 5'-CAGCATCGCAACCGCATCAG-3' (SEQ ID NO:6).
  • PCRkanHO has 145bp and 143bp homologous targeting sequences at the 5'- and 3'-ends of the kanamycin gene respectively.
  • PCR products were purified by gel electrophoresis, recovered through a QIAquick gel extraction kit (Qiagen, GmbH, Hilden, Germany) and quantitated.
  • EXAMPLE 7 Generation of recombinants
  • Fig. 5 The overall procedure for the generation of recombinants is outlined (Fig. 5). Frozen stocks of 40 ⁇ l of electrocompetent cells in microftige tubes were thawed on ice and 200ng of PCRkan50 or PCRkanHO was added into each tube of cells. The mixture of DNA and competent cells were transferred quickly into a pre-chilled 0.2cm electrode gap cuvette (BIO-RAD Labs., Hercules, CA) and electroporated using a BIO-RAD Gene Pulser apparatus. Electroporation conditions were: 2.5kV, 200 ⁇ , 25 ⁇ F.
  • LB medium was added to the cuvette immediately after electroporation, and the cells transferred into a 17x100mm FALCON polystyrene tube (BECKTON-DICKINSON Labware, Lincoln Park, NJ). The tubes were incubated in a 37°C shaker (220rpm) for 1.5 hours. Cells from each electroporation were spread onto LB plates containing 12.5 ⁇ g/ml chloramphenicol and 35 ⁇ g/ml kanamycin to identify recombinants.
  • Electroporation efficiency of each batch of competent cells was measured by an independent electroporation with lOng of pZeoSN2 (Invitrogen, Carlsbad, CA) and plating serial dilutions of the electroporation mixture onto low-salt LB (pH7.5) plates containing 25 ⁇ g/ml Zeocin. Total number of surviving cells after electroporation was measured by plating serial dilutions of the DH10B(pGETrec, pEBACHO) or DH10B(pGETrec, pEBAC/148) electroporated competent cells (without addition of D ⁇ A) onto LB plates containing lOO ⁇ g/ml ampicillin and 12.5 ⁇ g/ml chloramphenicol. Percentage of recombination was measured as percentage of kanamycin- and chloramphenicol-resistant colonies per survivors after electroporation.
  • D ⁇ A from chloramphenicol- and kanamycin-resistant colonies was isolated from 2ml overnight cultures grown in LB medium containing 12.5 ⁇ g/ml chloramphenicol and 35 ⁇ g/ml kanamycin using a modified alkaline lysis miniprep method.
  • 49 Notl- or Xhol- digested BAC D ⁇ A was analysed by pulsed field gel electrophoresis in a 1% (w/v) agarose gel (SeaKem, FMC Bioproducts, Rockland, ME), in 0.5x TBE buffer at 6V/cm and H°C.
  • the Low Range and Midrange I PFG markers (NEB, Inc., Beverly, MA) were used as standards.
  • PCR analysis of recombinant clones was performed directly on overnight cultures using the AmpliTaq DNA polymerase kit (Perkin Elmer Corp., Norwark, CT).
  • AmpliTaq DNA polymerase kit Perkin Elmer Corp., Norwark, CT.
  • One microlitre aliquots of recombinants from 2ml overnight cultures grown in the presence of 12.5 ⁇ g/ml chloramphenicol and 35 ⁇ g/ml kanamycin
  • the parent clone grown in the presence of 12.5 ⁇ g/ml chloramphenicol only
  • PCR primers used to verify pEBAC140::kanl40 and pEBAC/148::kanl40 were HOKA, 5'-ATAAGCTCATGGAGCGGCGTAAC-3' (SEQ ID NO:7) and 140KB, 5'-GTTCCACATTTCCATATAAAGGCCA-3' (SEQ ID NO:8).
  • PCR primers used to verify both pEBAC/148::kan50 and pEBAC140::kan50 were 140KF and 140KR (Fig.4).
  • PCR products were resolved on a 1.5% (w/v) agarose gel. PCR products were sequenced at the recombination junctions using the Thermo Sequenase radiolabel ed terminator cycle sequencing kit (Amersham Life Sciences, Inc., Cleveland, OH).
  • Vassaux G Manson AL and Huxley C. Copy number-dependent expression of a YAC-cloned human CFTR gene in a human epithelial cell line. Gene Ther 1997; 4: 618-623.
  • Gaensler KM Kitamura M and Kan YW. Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice. Proc Natl Acad Sci USA 1993; 90: 11381-11385.
  • Pavan WJ, Hieter P and Reeves RH Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome. Mol Cell Biol 1990; 10: 4163-4169.
  • Antoch MP et al. Functional identification of the mouse circadian Clock gene by transgenic BAC rescue. Cell 1997; 89: 655-667.
  • loannou P and DeJong P Construction of Bacterial Artificial Chromosome Libraries based on the modified PI (PAC) system.
  • PAC modified PI

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Abstract

L'invention concerne généralement une méthode de recombinaison d'au moins deux séquences nucléotidiques, et des agents utiles dans ladite méthode. L'invention porte, plus particulièrement, sur une méthode de recombinaison, dans une cellule hôte qui exprime la recBCD nucléase ou un dérivé fonctionnel de celle-ci, d'une séquence nucléotidique avec une séquence nucléotidique linéaire. La méthode de l'invention est utile, inter alia, pour la modification de chromosomes bactériens artificiels par recombinaison homologue avec une séquence d'ADN linéaire.
PCT/AU1999/000835 1998-10-30 1999-09-29 Methode de recombinaison et agents utiles dans ladite methode Ceased WO2000026396A1 (fr)

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WO2005010179A1 (fr) * 2003-07-24 2005-02-03 The University Of Hong Kong Procede de construction et de modification de grandes molecules d'adn
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CN102634534A (zh) * 2012-03-30 2012-08-15 深圳市中联生物科技开发有限公司 基于同源重组的核酸分子克隆方法及相关试剂盒
EP3160492A4 (fr) * 2014-06-30 2018-01-03 Murdoch Childrens Research Institute Agent thérapeutique helicobacter
CN105087517B (zh) * 2015-08-18 2019-05-07 翌圣生物科技（上海）有限公司 一种重组酶复合物及体外同源重组无缝克隆的方法

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US7517688B2 (en) 2000-02-25 2009-04-14 Stratagene California Method for ligating nucleic acids and molecular cloning
EP1178111A1 (fr) * 2000-08-03 2002-02-06 Lohmann Animal Health GmbH & Co. KG Vaccination contre des virus herpes associés aux cellules-hôtes
WO2002012288A3 (fr) * 2000-08-03 2002-04-11 Lohmann Animal Health Gmbh Vaccination contre l'herpesvirus associe a une cellule hote
EA010721B1 (ru) * 2000-08-03 2008-10-30 Ломанн Энимал Хелт Гмбх Унд Ко. Кг Вакцина против инфекции, вызванной вирусом герпеса, ассоциированным, по существу, с хозяйской клеткой
HRP20030147B1 (en) * 2000-08-03 2011-10-31 Lohmann Animal Health Gmbh & Co. Kg Vaccination aganist host cell-associated herpesviruses
CZ303904B6 (cs) * 2000-08-03 2013-06-19 Lohmann Animal Health Gmbh & Co. Kg Vakcína proti infekci zpusobené herpesvirem, rekombinantní virový genom a jejich pouzití
US9073979B2 (en) 2000-08-03 2015-07-07 Lohmann Animal Health Gmbh Vaccination against host cell-associated herpes viruses
WO2005010179A1 (fr) * 2003-07-24 2005-02-03 The University Of Hong Kong Procede de construction et de modification de grandes molecules d'adn
WO2011154927A3 (fr) * 2010-06-10 2012-02-16 Gene Bridges Gmbh Clonage direct
US10443051B2 (en) 2010-06-10 2019-10-15 Gene Bridges Gmbh Direct cloning
US12195732B2 (en) 2010-06-10 2025-01-14 Gene Bridges Gmbh Direct cloning

Also Published As

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EP1127152A1 (fr) 2001-08-29
JP2002528129A (ja) 2002-09-03
AUPP684998A0 (en) 1998-11-26
CN1331748A (zh) 2002-01-16
EP1127152A4 (fr) 2003-01-02

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