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Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac

a technology of eukaryotic transformation vector and eukaryotic transformation vector, applied in the field of transposon piggybac, can solve the problems of limited gene transfer length, complicated procedure, and limited effective size of genes that may be inserted, and achieve the effect of enhancing the infusion of dna molecules into cells

Inactive Publication Date: 2019-01-03
UNIV OF NOTRE DAME DU LAC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention identifies the specific sequences and configurations in the mobile genetic element piggyBac that are required for it to function as a transposon. This allows for easier and more efficient insertion of DNA molecules into cells. The invention also solves issues with using piggyBac for gene transfer by providing methods and compositions to enhance the size of genes that can be transferred. The vectors created using these methods may be used for producing transgenic organisms, and have been successfully tested in both plants and animals. Overall, the invention improves the use of piggyBac for gene transfer and provides valuable tools for research and development in this field.

Problems solved by technology

Although the reported piggyBac vector is useful, length of genes that could be transferred is limited by the size of the other components of the vector.
This size limited the effective size of genes that may be inserted, because plasmids larger than 10 KB are generally more difficult to construct, maintain, and transduce into host genomes.
Another problem was that previous cloning regimens involved the excision of a gene, the promoter controlling the gene, and polyadenylation signals, from one plasmid followed by insertion into the piggyBac transfer vector.
This procedure was often complicated by the lack of suitable restriction enzyme sites for these manipulations.

Method used

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  • Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
  • Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac
  • Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac

Examples

Experimental program
Comparison scheme
Effect test

example 2

istance Required Between Termini for Movement of a PiggyBac Transposon Construct

[0109]The interplasmid transposition assay was carried out essentially as previously described by Lobo et al. (1999), Thibault et al. (1999) and Sarkar et al. (1997a). Embryos were injected with a combination of 3 plasmids. The donor plasmid, pB(KOα), carried a piggyBac element marked with the kanamycin resistance gene, ColE1 origin of replication, and the lacZ gene. The transposase providing helper plasmid, pCaSpeR-pB-orf, expressed the full length of the piggyBac ORF under the control of the D. melanogaster hsp70 promoter. The target B. subtilis plasmid, pGDV1, is incapable of replication in E. coli, and contains the chloramphenicol resistance gene. Upon transposition of the genetically tagged piggyBac element from pB(KOα) into the target plasmid pGDV1 with the help of the transposase provided by the helper pCaSpeR-pB-orf that expresses the piggyBac transposase protein from a minimal hsp70 promoter (se...

example 3

mid Transposition Assay of pCRII-ITR and pBSII-ITR Plasmids

[0112]According to an embodiment of the present invention, the excision assay described herein shows that a minimum of 163 bp of the 3′ terminal region and 125 bp of the 5′ terminal region (from the restriction site SacI to the end of the element) may be used for excision, while the pIAO-P / L constructs showed that a minimal distance of 55 bp between termini may be utilized to effect movement. These data suggested that the inclusion of intact left and right terminal and internal repeats and spacer domains would be sufficient for transposition.

[0113]The pCRII-ITR plasmid was constructed following PCR of the terminal domains from pIAO-P / L-589 using a single IR specific primer. A second construct pCRII-JFO3 / 04 was also prepared using two primers that annealed to the piggyBac 5′ and 3′ internal domains respectively, in case repeat proximate sequences were required.

[0114]The interplasmid transposition assay was performed in T. ni ...

example 4

ion of Minimum PiggyBac Vector pXL-Bac

[0116]A new piggyBac minimum vector pXL-Bac (FIG. 3(C2)) was also constructed by combining the 702 bp BamHI ITR fragment with the pBlueScript II BamHI fragment and inserting a PCR amplified pBSII multiple cloning site (MCS) between the terminal repeats. The pXL-Bac vector was tested by inserting an XbaI fragment from πKOα (obtained from A, Sarkar, University of Notre Dame), containing the Kanamycin resistance gene, E. coli replication origin, and Lac a-peptide, into the MCS of pXL-Bac to form pXL-Bac-KOa. Interplasmid transposition assays yielded a frequency of over 10−4 for transposition of the modified ITR sequence, a similar level as observed for the intact piggyBac element.

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Abstract

Isolated nucleic acid molecules that include a minimally-sized, functional (minimally-functional) piggyBac transposon can incorporate (i) a 5′ internal domain (ID) comprising a nucleotide fragment that is substantially homologous to a native piggyBac transposon sequence; (ii) a 5′ terminal repeat domain (TRD) comprising a 5′ terminal repeat (TR) sequence, a 5′ spacer sequence, and a 5′ internal repeat (IR) sequence; (ii) a sequence of interest; (iv) a 3′ TRD comprising a 3′ IR sequence, a 3′ spacer sequence, and a 3′ TR sequence; and (iv) a 3′ ID comprising a nucleotide fragment that is substantially homologous to the native piggyBac transposon sequence. The 5′ TRD and the 3′ TRD can be optionally linked by a sequence comprising a multiple cloning site.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 11 / 454,947 filed on Jun. 19, 2006, entitled “METHODS AND COMPOSITIONS FOR TRANSPOSITION USING MINIMAL SEGMENTS OF THE EUKARYOTIC TRANSFORMATION VECTOR PIGGYBAC,” which is a continuation-in-part of U.S. patent application Ser. No. 10 / 826,523 filed on Apr. 19, 2004, entitled “METHODS AND COMPOSITIONS FOR TRANSPOSITION USING MINIMAL SEGMENTS OF THE EUKARYOTIC TRANSFORMATION VECTOR PIGGYBAC,” which issued as U.S. Pat. No. 7,105,343 on Sep. 12, 2006, which is a continuation-in-part of U.S. patent application Ser. No. 10 / 001,189 filed on Oct. 30, 2001, entitled “METHODS AND COMPOSITIONS FOR TRANSPOSITION USING MINIMAL SEGMENTS OF THE EUKARYOTIC TRANSFORMATION VECTOR PIGGYBAC,” which issued as U.S. Pat. No. 6,962,810 on Nov. 8, 2005, which claims benefit of and priority to U.S. Provisional Patent Application No. 60 / 244,984 filed on Nov. 1, 2000 entitled “Methods and compositi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/85C12N15/90
CPCC12N2800/90C12N15/85C12N15/90C12N2800/204
Inventor FRASER, MALCOLMLI, XU
Owner UNIV OF NOTRE DAME DU LAC
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