| US 7,592,161 B2 | ||
| Methods for analyzing the insertion capabilities of modified group II introns | ||
| Alan M. Lambowitz, Austin, Tex. (US); Huatao Guo, Alhambra, Calif. (US); and Michael Karberg, Austin, Tex. (US) | ||
| Assigned to The Ohio State University Research Foundation, Columbus, Ohio (US); and The University of Texas Board of Regents of the University of Texas System, Austin, Tex. (US) | ||
| Filed on Oct. 22, 2002, as Appl. No. 10/277,643. | ||
| Application 10/277643 is a continuation of application No. 09/687944, filed on Oct. 13, 2000, abandoned. | ||
| Claims priority of provisional application 60/159724, filed on Oct. 15, 1999. | ||
| Prior Publication US 2003/0104352 A1, Jun. 05, 2003 | ||
| Int. Cl. C12P 19/34 (2006.01); C12P 12/06 (2006.01) | ||
| U.S. Cl. 435—91.1 [435/69.1; 435/194; 536/23.2; 536/23.4; 536/24.1] | 18 Claims |
| 1. A method of increasing insertion of modified group II introns into a DNA target site in a host cell in vitro, comprising:
a) introducing a nucleic acid construct into the host cell, said construct comprising:
i.) a modified group II intron sequence encoding a modified group II intron RNA, said modified group II intron RNA comprising
an EBS1 sequence, an EBS2 sequence, a delta sequence, and a deletion of subdomains IVB1, IVB2, IVB3, wherein one or more of
said EBS1, EBS2 or delta sequences is modified to provide a modified EBS1, EBS2, and delta sequence that base pairs with an
IBS1, IBS2, and delta prime sequence, respectively, in the DNA target site;
ii) a promoter for regulating transcription of said modified group II intron sequence, said promoter being operably linked
to the modified group II intron sequence;
iii) a first flanking sequence upstream of the modified group II intron sequence, said flanking sequence encoding a first
hybridizing sequence which is complementary to the EBS1 or the modified EBS1 sequence contained in said modified group II
intron RNA, and a second flanking sequence downstream of the modified group II intron sequence encoding a second hybridizing
sequence which is complementary to the EBS2 or the modified EBS2 sequence contained in said modified group II intron RNA;
and
iv) an open reading frame sequence encoding a wild-type or modified group II intron encoded protein,
wherein said open reading frame sequence is located upstream, or downstream of the modified group II intron sequence, and
wherein expression of said group II intron encoded protein is regulated by the promoter which is operably linked to the modified
group II intron sequence or by a second promoter which is operably linked to the open reading frame sequence; and
b) maintaining the cell under in vitro conditions which allow for expression of the modified group II intron sequence and
the open reading frame sequence, formation of RNP particles comprising a modified excised group II intron RNA which is encoded
by the modified group II intron sequence and a group II intron encoded protein which is encoded by the open reading frame
sequence, and insertion of the modified group II intron into the DNA target site;
wherein the method results in increased insertion of said modified group II introns into the DNA target site in the host cell
in vitro as compared to a method where the group II intron RNA lacks deletion of subdomains IVB1, IVB2, and IVB3.
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