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Transposon-based transformation vectors
| Details |
Inventors: Cooper, Richard K.;
Assignee: Board of Supervisors of Louisiana State University and Agricultural and (Baton Rouge, LA)
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Nelson; Amy J.
Attorney, Agent or Firm: Runnels; John H.
A novel transposen-based vector has been developed that enhances the integration of DNA into a host genome, particularly a eukaryotic genome. The novel vector has been used, for example, in the transformation of mammalian and fish cells with a gene coding for the inductive expression of the lytic peptide cecropin B, and in the production of transgenic fish containing a gene coding for the inductive expression of the lytic peptide cecropin B. This vector shows greater transformation efficiencies than do other means for transforming a gene into a eukaryotic chromosome. The novel vector allows the rapid and efficient transformation of a eukaryotic genome. Its use does not require the high level of skill needed for microinjections. Nor does it rely on homologous recombination events for a successful transformation, as do the prior methods of microinjection, electropotation, and lipofection. A novel modification of mini-transposons was made to adapt them to carry a gene of interest into a genome. |
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DETAILED DESCRIPTION I claim: 1.
A vector for inserting an exogenous gene into a chromosome of a eukaryotic cell, comprising: (a) a gene encoding a bacterial transposase; (b) two transposon insertion sequences recognized by the transposase; (c) said exogenous gene, wherein said exogenous gene is between the two transposon insertion sequences; and (d) a promoter that is operably linked to said transposase gene; wherein one of said insertion sequences is located between said transposase gene and said exogenous gene; and wherein the transposase expressed by said transposase gene will excise from said vector a fragment comprising the two transposon insertion sequences and the exogenous gene between the two transposon insertion sequences, and will insert the excised fragment into a chromosome of a eukaryotic cell.
2.
A vector as recited in claim 1, wherein said promoter is inducible by an inducing stimulus to cause the transcription of said transposase.
3.
A vector as recited in claim 2, wherein said vector is plasmid pCEP.
4.
A vector as recited in claim 3, wherein said exogenous gene is a gene encoding native cecropin B.
5.
A vector as recited in claim 3, wherein said exogenous gene is a gene that is not a gene encoding native cecropin B.
6.
A cell from the E.
coli strain with ATCC accession number 69342.
Description:
This invention pertains to transposon-based transformation vectors.
Transformation of Eukaryotic Genomes A major problem in transforming DNA into eukaryotic cells, especially into the cells of mammals and other vertebrates, is the stable integration of the exogenous DNA into a recipient chromosome.
Several techniques are currently used for the delivery of the DNA into the recipient host, techniques that have shown varying degrees of success.
The first, and currently most common, method for transformation of animals is the microinjection of exogenous DNA into a one- or two-cell embryo.
This procedure has several drawbacks, including the following difficulties: (1) the technique requires a level of skill that is not available in most laboratories; (2) the procedure is very time-consuming, often requiring an entire day to microinject a few hundred embryos; and (3) the method has a relatively low success rate--typically about 1-3% of the injected embryos are observed to have a stable insertion
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