Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Vectors comprising novel regulatory elements

一种载体、元件的技术，应用在重组DNA领域，能够解决5-甲基胞嘧啶不稳定等问题

Inactive Publication Date: 2008-02-06
EMD MILLIPORE CORP
View PDF9 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 5-methylcytosine is unstable and converts to thymine

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Vectors comprising novel regulatory elements
  • Vectors comprising novel regulatory elements
  • Vectors comprising novel regulatory elements

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Generation of stably transfected CHO-K1 cells using vectors including hCMV promoter or gpCMV promoter

[0078] Plasmid constructs were prepared as follows. Ampicillin resistance gene by PCR from pBluescript  (Stratagene) isolation, introducing Nru I sites at each end of primers 5'-TGTCGCGAGTCTGACAGTTACCAATGCTTAATC3' (SEQ ID NO: 5), 5'-CATCGCGAGCACTTTTCGGGGAAAT GTGTGCGC-3' (SEQ ID NO: 6). The PCR product was inserted into the Pvu II site of pMae II (Nucleic Acids Research 2001 29:E26) to generate pCA1. The following oligonucleotides

[0079] 1.5′-TCGAAGTTTAAACATTTAAATCTAGAAGCTTAT-3′

[0080] (SEQ ID NO: 7)

[0081] 2.5′-CCGGTATCGATAAGCTTCTAGATTTAAATGTTTAAACT-3′

[0082] (SEQ ID NO: 8)

[0083] 3.5'-CGATACCGGTGGCGCGCCAATTGTTAATTAAGATCTGG-3'

[0084] (SEQ ID NO: 9)

[0085] 4.5'-CCCATTGGGCCAGATCTTAATTAACAATTGGCGCGCCA-3'

[0086] (SEQ ID NO: 10)

[0087] 5.5'-CCCAATGGGCCGTACGAATTCCTTAGGCTCGAG-3'

[0088] (SEQ ID NO: 11)

[0089] 6.5'-GGCCCTCGAGCCTAAGGAATTCGTACG...

Embodiment 2

[0095] HEK293 cells were cultured in Dulbecco's Eagle medium (DMEM; Sigma, UK) supplemented with 10% fetal bovine serum and 5 U / ml penicillin and streptomycin mixture (Sigma, UK). For stable transfection, HEK293 cells were treated with 1×10 6 Cells / well density were seeded in 6-well plates and then incubated at 37°C in 5% CO 2 Incubate for 24 hours in the incubator. Cells were then transfected with 4 μg of the indicated plasmids (pCET1005-EGFP or pCET1005-gpCMV-EGFP) (linearized with Pci I) with 10 μl Lipofectamine 2000 (Invitrogen, UK).

[0096] DNA and Lipofectamine 2000 were each diluted in 250 μl of OptiMEM I (Gibco, UK), incubated at room temperature for 5 minutes, mixed and then incubated for another 20 minutes. The growth medium of the cells was replaced with 1 ml OptiMEM I supplemented with 15% FCS, followed by the DNA / Lipofectamine 2000 mixture. Cells were incubated at 37°C in 5% CO before adding 3.5 ml of OptiMEMI supplemented with 10% FCS 2 Incubate for 5 hours ...

Embodiment 3

[0098] CHO-K1 cells were cultured as described in Example 1. Inoculate 1.5×10 cells 24 hours before transferring to 12 wells 5CHO-K1 cells. After 24 hours, cells were transfected with 1 μg luciferase reporter plasmid (phCMV-Luc or pgpCMV-Luc) with 1.5 μl FUGENE (Roche, UK). For this, FUGENE and DNA were diluted separately into Opti-MEM I (Invitrogen), mixed, and incubated for 30 minutes at room temperature before adding to the cells. Luciferase expression was analyzed after 24 hours using a Berthold luminometer (Berthold, Wildbad, Germany). Typically, cell lysis and luciferase reporter assays were performed as previously described (Lipinski et al., Gene Therapy, 2001(8):274-281). Transfections were performed in triplicate and the mean and standard deviation for each representative experiment are shown (Figure 7). It is clear that the luciferase activity of the gpCMV vector is at least 2-fold higher than that of the hCMV plasmid.

[0099] The plasmid hCMV-Luc has been desc...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the field of recombinant DNA technology and, in particular, the development of vectors for the expression of recombinant proteins. Expression of heterologous genes in eukaryotic cells requires transcription by RNA polymerase II, which is driven by c / s-acting genetic elements known as promoters and enhancers. The invention provides promoters derived from the guinea pig cytomegalovirus early- immediate promoter / enhancer, useful for obtaining high levels of expression of recombinant proteins. Also disclosed are eukaryotic expression vectors comprising such promoters, which are capable of providing increased levels of expression over that obtainable from human or murine cytomegalovirus enhancer / promoter elements in many cell types.

Description

Background technique [0001] The invention relates to the technical field of recombinant DNA, especially the vector developed for expression of recombinant protein. Expression of heterologous genes in eukaryotic cells is an important aspect of biotechnology with both academic and commercial applications. Expression of these genes requires RNA polymerase II (PolII) for transcription, which is driven by cis-genetic elements known as promoters and enhancers. [0002] Briefly, a promoter is a directional element for transcription of an initiation sequence located less than 100 (usually less than 50) base pairs (bp) downstream. They contain many short consensus nucleotide sequences that serve as binding sites for different proteins involved in transcription initiation and assembly of a multi-subunit complex known as the pre-initiation complex (McKnight and Tjian, 1987 , Cell 46:795-805). In most genes this occurs at a very widely conserved sequence known as the TATA box (TATAAA),...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2830/46C12N2710/16122C07K14/005
Inventor 史蒂文·杰兰特·威廉姆斯乔纳森·高恩阿利斯泰尔·辛普森·欧文
Owner EMD MILLIPORE CORP
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com