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Circular nucleic acid vectors, and methods for making and using the same

A circular, vector technology, applied in the field of molecular biology, can solve the problems of slow recombination process, immunogenic integration mutation, etc.

Inactive Publication Date: 2007-04-11
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of techniques that rely on homologous recombination are: the homologous sequences necessary for homologous recombination to occur may not necessarily exist; the recombination process may be slow, etc.
Defects associated with viral vectors include immunogenicity, virus-induced complications, and integration-induced mutational issues

Method used

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  • Circular nucleic acid vectors, and methods for making and using the same
  • Circular nucleic acid vectors, and methods for making and using the same
  • Circular nucleic acid vectors, and methods for making and using the same

Examples

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preparation example Construction

[0066] Preparation method of minicircle carrier

[0067] As outlined, the present invention also provides methods for efficiently preparing the minicircle vectors of the present invention. When the minicircle vector is prepared according to a specific embodiment of the present invention, the target expression cassette is contained, and the expression cassette is flanked by the parent nucleic acid of attB and attP sites and the unidirectional site-specific recombinase capable of recognizing attB and attP sites in a suitable Contacting under conditions, so that recombination is mediated by a unidirectional site-specific recombinase, so that the minicircle vector of the present invention is produced from the parental nucleic acid. "Both sides" refers to the expression cassette sequence, or other target sequences to be included in the minicircle vector, and each has an att site at its two ends, such as attB and attP. Therefore, the parental nucleic acid is represented by the follo...

Embodiment

[0082] I. Materials and methods

[0083] A. Construction of the carrier:

[0084]To make the hAAT minicircle construct pBAD.C31.RHB (Fig. 1a), we amplified the C31 integrase from pCMV.C31 (Groth, A.C., Olivares, E.C., Thyagarajan, B. & Calos, M.P. Aphage Integrase directs efficient site-specific integration in human cells.Proc NatlAcad SCI USA 97,5995-6000.(2000)), the primers used are: 5'-CCG TCC ATG GACACG TAC GCG GGT GCT (SEQ ID NO: 01) and 5′-ATG CGC GAG CTC GGT GTCTCG CTA CGC CGC TAC (SEQ ID NO: 02), the PCR product was inserted into the Nco I and Sac I sites of pBAD / Myc-His (Invitrogen, Carlsbad, CA) to obtain the intermediate plasmid pBAD .C31. We synthesized attB and attP using the corresponding DNA oligonucleotides (Groth et al., supra) and inserted them into the Spe I and Kpn I sites flanking the hAAT expression cassette on plasmid pRSV.hAAT.bpA, respectively (Fig. 2a). The attB, aatP binding sites and the expression cassette were inserted together between the...

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Abstract

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits / systems for practicing the same.

Description

[0001] related application [0002] This patent application claims priority to U.S. Provisional Patent Application No. 60 / 407,344, filed August 29, 2002, and U.S. Provisional Patent Application No. 60 / 463,672, dated April 16, 2003; the contents of which are incorporated herein among. field of invention [0003] The present invention relates to molecular biology and, more particularly, to transformation and especially vectors for transformation. Background of the invention [0004] The process of introducing exogenous nucleic acid sequences (such as DNA) into cells is called "transformation" and plays an important role in various biotechnology and related application fields including research, synthesis and application. Transformation plays a key role in research applications, including the creation of transgenic cells and transgenic animals. The key role transformation plays in synthetic applications includes the production of peptides and proteins,...

Claims

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Application Information

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IPC IPC(8): C12N15/02C12N15/00
Inventor M·A·凯Z·－Y·陈
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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