Patent ID: 6485912
Filing Date: 2002-11-26
Classification: A01K,C12N

Abstract:
A method of placing a eukaryotic promoter exon/intron unit (PEU) into a Bacterial or Bacteriophage-Derived Artificial Chromosome (BBPAC) that contains a trappable eukaryotic gene comprising:(a) introducing a conditional replication shuttle vector into a host cell containing the BBPAC under conditions in which the conditional replication shuttle vector can replicate and transform the host cell, wherein: (i) the BBPAC contains a trappable eukaryotic gene, BBPAC vector DNA, and a second marker gene; (ii) the conditional replication shuttle vector contains a first marker gene which can be counter-selected against; a recombination cassette; and a gene encoding a protein that supports homologous recombination, wherein said protein remedies a deficiency of an endogenous recombination protein in said host cell; wherein the recombination cassette comprises a PEU flanked on both its 5â€² and 3â€² ends by nucleotide sequences that are homologous to BBPAC vector DNA; wherein the recombination cassette, the gene encoding said protein and the first marker gene are linked together on the conditional replication shuttle vector such that when the PEU integrates into the BBPAC, the gene encoding'said protein and the first marker gene remain linked together, but neither the gene encoding said protein nor the first marker gene remain linked to the integrated PEU; (iii) the PEU comprises a third marker gene, a eukaryotic promoter, at least one 5â€² vector-derived exon, and an intron or fragment thereof; wherein the 5â€² vector-derived exon is adjacent to the intron or fragment thereof and is operatively downstream from the eukaryotic promoter; wherein when the trappable eukaryotic gene comprises an exon with a 3â€² splice acceptor, the PEU can integrate into the BBPAC placing the exon of the trappable eukaryotic gene operatively downstream of the PEU; and (iv) wherein neither the host cell nor the BBPAC independently or in conjunction can support homologous recombination, without the conditional replication shuttle vector; (b) growing the host cell under conditions in which the conditional replication shuttle vector can replicate, the gene encoding said protein can be expressed, and in which a cell that contains the first and second marker genes is selected for; and wherein a first homologous recombination event occurs between the recombination cassette and the BBPAC to form a co-integrate; (c) growing the cell selected for in step (b) under conditions in which the conditional replication shuttle vector cannot replicate and in which a cell that contains the first and second marker proteins is selected for, whereby a cell containing the co-integrate between the recombination cassette and the BBPAC is selected for; and (d) growing the cell selected for in step (c) under conditions in which the conditional replication shuttle vector cannot replicate and in which a cell that contains the second marker gene is selected for; wherein a second homologous recombination event occurs between the conditional replication shuttle vector and the BBPAC; and wherein the PEU is placed into the BBPAC.