Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-03-08
2003-04-15
Ketter, James (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
Reexamination Certificate
active
06548736
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to gene targeting.
Gene targeting is a process whereby a specific gene, or a fragment of that gene, is altered. This alteration of the targeted gene may result in a change in the level of RNA or protein that is encoded by that gene, or the alteration may result in the targeted gene encoding a different RNA or protein than the untargeted gene. The targeted gene may be studied in the context of a cell, or, more preferably, in the context of a transgenic animal.
Transgenic animals are among the most useful research tools in the biological sciences. These animals have a heterologous (i.e., foreign) gene, or gene fragment, incorporated into their genome that is passed on to their offspring. Although there are several methods of producing transgenic animals, the most widely used is microinjection of DNA into single cell embryos. These embryos are then transferred into pseudopregnant recipient foster mothers. The offspring are then screened for the presence of the new gene, or gene fragment. Potential applications for transgenic animals include discovering the genetic basis of human and animal diseases, generating disease resistance in humans and animals, gene therapy, drug testing, and production of improved agricultural livestock.
SUMMARY OF THE INVENTION
In general, the invention features methods and uses for transposon-mediated gene targeting which greatly enhance the insertion and detection of desired genes in genomic exons by homologous recombination. The invention also features diagnostic methods for endocrine disorders, as well as methods and reagents for treating endocrine disorders.
In a first aspect, the invention provides a method for targeting heterologous DNA to integrate into an exon of a eukaryotic cell. The method includes, first, generating a pool of bacteria containing plasmids into which have been randomly integrated a transposon including heterologous DNA; second, isolating from the pool a bacterium which contains a plasmid into which the transposon is integrated into a copy of the exon on the plasmid by assessing PCR amplification products generated from the pool using primers specific for the exon; third, introducing the plasmid of the bacteria into the cell under conditions that promote homologous recombination; and, fourth, screening genomic DNA of the cell for integration of the heterologous DNA into the exon of the cell.
In one embodiment of the first aspect of the invention, the transposon bears at its extremities recognition sequences of a first rare-cutting restriction endonuclease that is absent in the exon. In another embodiment, the heterologous DNA, or portion thereof, encodes a selectable marker protein. The heterologous DNA, or portion thereof, may additionally encode a second protein, or polypeptide fragment thereof. In another embodiment, the marker protein is a prokaryotic selectable marker protein, which may be replaced by a eukaryotic selectable marker protein via the recognition sequences of the first rare-cutting restriction endonuclease. The prokaryotic selectable marker protein may be additionally replaced with DNA, or a portion thereof, encoding a second protein, or polypeptide fragment thereof.
In another embodiment of this aspect, the exon copy or portion thereof has at its borders destroyed recognition sequences of a second rare-cutting restriction endonuclease. In another embodiment, the genomic DNA is digested with the second rare-cutting restriction endonuclease. In yet another embodiment, the screening is carried out by Southern blot analysis of the genomic DNA with a detectable probe specific for the exon, or with a detectable probe external to the exon. The screening may also be carried out by PCR amplification of the genomic DNA with primers specific for the exon, or with primers external to, but surrounding the exon such that the PCR product includes the exon.
In a preferred embodiment of the first aspect of the invention, the insertion of the heterologous DNA into the exon results in a reduced level of expression of the protein encoded by the gene of the exon. The insertion of the heterologous DNA into the exon may also result in the expression of a truncated protein encoded by the gene of the exon, expression of a fusion protein encoded by the gene of the exon and the heterologous DNA, or portion thereof, or expression of a product, which may be a fusion protein, encoded by the heterologous DNA, or portion thereof.
In a second aspect, the invention provides a method for making a transgenic, non-human vertebrate animal containing heterologous DNA by first producing an embryonal cell of the non-human vertebrate animal with a targeted exon by first, generating a pool of bacteria containing plasmids into which have been randomly integrated a transposon including heterologous DNA; second, isolating from the pool a bacterium which contains a plasmid into which the transposon is integrated into a copy of the exon on the plasmid by assessing PCR amplification products generated from the pool using primers specific for the exon; third, introducing the plasmid of the bacteria into the embryonal cells under conditions that promote homologous recombination; and fourth, screening genomic DNA of the embryonal cells to identify an embryonal cell in which there has occurred integration of the heterologous DNA into the exon. The identified embryonal cell is then grown to generate the transgenic animal.
In one embodiment of the second aspect of the invention, the transposon bears at its extremities recognition sequences of a first rare-cutting restriction endonuclease that are absent in the exon. In another embodiment, the heterologous DNA, or portion thereof, encodes a selectable marker protein. The heterologous DNA, or portion thereof, additionally encodes a second protein, or polypeptide fragment thereof.
In another embodiment, the marker protein is a prokaryotic selectable marker protein which may be replaced by a eukaryotic selectable marker protein via the recognition sequences of the first rare-cutting restriction endonuclease. In another embodiment, the prokaryotic selectable marker protein is additionally replaced with DNA, or a portion thereof, encoding a second protein, or polypeptide fragment thereof.
In another embodiment, the exon copy or portion thereof has at its borders destroyed recognition sequences of a second rare cutting restriction endonuclease. Genomic DNA may be digested with the second rare-cutting restriction endonuclease. In another embodiment, the screening is carried out by Southern blot analysis of the genomic DNA with a detectable probe specific for the exon, or with a detectable probe external to the exon. The screening may also be carried out by PCR amplification of the genomic DNA with primers specific for the exon, or with primers external to, but surrounding the exon such that the PCR product includes the exon.
In a preferred embodiment of this aspect of the invention, the animal expresses a reduced level of the protein encoded by the gene of the exon. In another embodiment, the animal expresses a truncated protein encoded by the gene of the exon. In another embodiment, the animal expresses a fusion protein product encoded by the gene of the exon and the heterologous DNA, or portion thereof. In another embodiment, the animal expresses a product, which may be a fusion protein, encoded by the heterologous DNA, or portion thereof.
In a third aspect, the invention features a transposon that includes a selectable marker cassette including the selectable marker operably linked to a promoter, or hybrid thereof, capable of expressing the marker in both eukaryotic and prokaryotic cells. In a preferred embodiment of this aspect of the invention, the selectable marker is both a prokaryotic and eukaryotic selectable marker. In another embodiment of this aspect of the invention, the cassette is flanked by the recognition sequences of one or more rare-cutting restriction endonucleases. Most preferably, the transposon of this aspect of the invention is used to integrate a
Leder Philip
Lindberg Iris
Westphal Christoph H.
Ketter James
President and Fellows of Harvard College
Ropes & Gray
Schnizer Richard
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