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Improved genetic elements providing high levels of expression

A technology of polynucleotides and vectors, applied in the field of polynucleotides, can solve problems such as instability

Active Publication Date: 2006-03-15
MILLIPORE CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 5-methylcytosine is unstable and is converted to thymine

Method used

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  • Improved genetic elements providing high levels of expression
  • Improved genetic elements providing high levels of expression
  • Improved genetic elements providing high levels of expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0096] Example 1A Enhanced expression of 1.5kb HP-1 / hnRNP A2UCOE

[0097] Materials and methods

[0098] Vector construction

[0099] As described in our prior application WO 00 / 05393, the vector CET20 was obtained by cloning the 8.3 kb HindIII fragment of the human HP-1 / hnRNP A2 locus (which contains the HP1 / RNP promoter and extended CpG island) into generated in pBluescript (Stratagene).

[0100] The inserted 4186bp (referred to as 4kb) fragment was then removed by BamHI and HindIII digestion. These fragments were blunt-ended using T4 DNA polymerase and ligated into pEGFPN-1 (Clontech) which had been digested with Asel and blunt-ended again using T4 DNA polymerase. Clones with fragments in both orientations were then isolated.

[0101] A 1546bp Esp31 (isoschizase of BsmBI) fragment (referred to as a 1.5kb fragment) was again isolated from CET20 by Esp31(BsmBI) digestion followed by blunt-end filling, and these were then ligated into Asel of pEGFPN-1 as described above ...

Embodiment 2A

[0109] Example 2A 1kb HP-1 / hnRNP A2 UCOE enhanced expression

[0110] Materials and methods

[0111] Vector construction

[0112] The vector containing 1 kb UCOE was filled in and religated by digesting the pEGFPN-1 vector with the 1.5 kb Esp31 fragment in the forward direction with PciI and BspEI to remove 5'500 bp. This resulted in a vector with the 987bp BspEl-Esp31 fragment in only one orientation.

[0113] result

[0114] The forward 1kb (987bp) fragment showed a fluorescence median value comparable to that of the forward 1.5kb fragment ( Figure 5 ) and positive cells % ( Image 6 )quite

Embodiment 3A

[0115] Example 3A 1.5kb HP-1 / hnRNP A2 UCOE enhances adenovirus-encoded constructs Express

[0116] Materials and methods

[0117] cell culture

[0118] HeLa was obtained from ATCC (Manassas, Virginia). PER.C6 was obtained from Crucell, (Leiden, The Netherlands). All purchased cell lines were cultured as recommended by the manufacturer. 911 cells were a kind gift from Prof. L.S. Young (Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK) and cultured in DMEM / 10% FCS containing antibiotics.

[0119] Plasmid construction

[0120] PGL3basic was obtained from Promega (Madison, WI, USA) and contained the luciferase-SV40p (A) cassette downstream of the multiple cloning site. The human CMV enhancer / promoter (0.9 kb) was cloned into Smal digested pGL3basic to make pGL3 / CMV-Luc-SV40p (A). To prepare pGL3 / 1.5kb(F)UCOE-CMV-SV40p(A), the 1.5kb UCOE Esp3I fragment (see figure 1 and 2 ) was blunt-ended with T4 DNA polymerase (NEB, Beverl...

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Abstract

The invention relates to improved genetic elements providing high levels of expression of operably-linked genes in a variety of tissues. In particular, fragments of unmethylated, CpG islands of less than 2kb are shown to provide enhanced transgene expression and have advantages in terms of vector construction and cloning capacity.

Description

field of invention [0001] The present invention relates to polynucleotides comprising improved, smaller ubiquitous chromatin opening elements (UCOEs). When operably linked to an expressible nucleic acid sequence, the elements produce high and reproducible levels of gene expression. The present invention also relates to vectors containing said polynucleotide sequences, host cells containing said vectors and the use of said polynucleotides, vectors or host cells in therapy, or for expressing proteins in cell culture. Background of the invention [0002] Current models of chromatin structure in higher eukaryotes assume gene organization in a "domain format" (Dillon, N. & Grosveld, F. Chromatin Domains as Potential Eukaryotic Gene Functional Units, Curr. Opin. Genet. Dev 4, 260-264 (1994); Higgs, D.R. Open chromatin domains of LCRs? Cell 95, 299-302 (1998)). Chromatin domains are imagined to exist in a condensed, 'closed', transcriptionally silent state, or a decondensed, 'ope...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12Q1/68A61K48/00
Inventor 史蒂文·杰兰特·威廉姆斯罗伯特·拉克兰·克龙比凯·史蒂文·利平斯基阿利斯泰尔·辛普森·欧文
Owner MILLIPORE CORP
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