Tissue specific expression of retinoblastoma protein

A technology for expressing vectors and fusion polypeptides, applied in the direction of retinoblastoma protein, mammalian protein, peptide/protein components, etc., which can solve the problems of cell growth stagnation and unclear exact pathway of arrest

Inactive Publication Date: 2007-09-19
CANJI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RB arrests cell growth by blocking the exit from G phase into S phase (e.g., Dowdy et al. Cell 73:499-511(1993)), but the exact pathway of its arrest is still unclear

Method used

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  • Tissue specific expression of retinoblastoma protein
  • Tissue specific expression of retinoblastoma protein
  • Tissue specific expression of retinoblastoma protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0081] E2F-RB fusion

[0082] A. Introduction

[0083] In this example, expression plasmids encoding different segments of E2F fused to RB56 polypeptides were constructed. RB56 is a subfragment of full-length RB that contains the "pocket" domain necessary for growth inhibition (Hiebert et al. MCB 13:3384-3391 (1993); Qin et al. Genes and Rev. .6: 953-964 (1992)). E2F194 comprises E2F amino acids 95-194. This fragment contains only the DNA-binding domain of E2F. E2F286 contains a DNA binding domain and a DP-1 heterodimerization domain. Both E2F fragments lack the N-terminal cyclin A-kinase-binding domain, which appears to downregulate the DNA-binding activity of E2F (Krek et al. Cell 83:1149-1158 (1995); Krek et al., Cell 78:161-172 (1994)).

[0084] B. Construction of the vector

[0085] Plasmid pCTM contains a CMV promoter, a triple adenoviral leader flanked by T7 and SP6 promoters, a multiple cloning site, and downstream of the multipl...

Embodiment II

[0108] Tissue-specific expression of E2F-RB fusions

[0109] A. Construction of recombinant adenovirus

[0110] In this experiment, recombinant adenoviruses containing the RB polypeptide under the control of a CMV promoter or the smooth muscle alpha-actin promoter were generated.

[0111]Isolation of the smooth muscle α-actin promoter (bases -670 to +5) by PCR from a genomic library, Reddy et al., "Construction of the human smooth muscle α-actin gene" J. Biol. Chems .265: 1683-1687 (1990), Nakano et al., "Transcriptional regulatory elements and the first intron 5' upstream of the human smooth muscle (aortic type) α-actin-encoding gene" Gene 99:285-289 (1991)), and added 5' Xho I and Avr II and 3' Xha I, Cla I and Hind III restriction enzyme sites for cloning purposes. The fragment was subcloned into a plasmid as an Xho I, Hind III fragment and sequenced to confirm its base composition. A fusion construct 286-56 was subcloned as an Xha I, Cla I fragment di...

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Abstract

Fusions of the transcription factor E2F and the retinoblastoma protein RB are provided, along with methods of treatment of hyperproliferative diseases.

Description

Background of the invention [0001] Both the retinoblastoma gene (RB) and the transcription factor E2F have key roles in cell growth control (see Adams, P. and Kaelin, W. Seminars in Cancer Biology 6:99-108 (1995) for a review). The RB locus is frequently inactivated in various human tumor cells. The wild-type RB gene (as in Bookstein et al., Science 247:712-715 (1990)) or wild-type RB protein (pRB) (such as Antelman et al., Oncogene 10:697-704 (1995)) reintroduction of RB-negative / RB mutant cells can inhibit the growth of these cells in culture as well as their tumorigenic capacity in vivo. [0002] The role of E2F is to activate the transcription of S phase genes, while RB represses its activity. RB arrests cell growth by blocking the exit from G phase into S phase (e.g., Dowdy et al. Cell 73:499-511 (1993)), but the exact pathway of its arrest is still unclear. [0003] Although E2F forms a complex with RB, complex formation is more efficient if an E2F-associated ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/09C07K19/00C07H21/04C07K5/00A61K38/00A61K35/12A61P35/00A61K31/7088A61K48/00A61P5/00A61P13/08A61P17/06A61P27/02A61P43/00C07K14/47C07K14/82
CPCA61K48/00C12N2799/022C07K2319/00C07K14/4736A61P13/08A61P17/06A61P27/02A61P35/00A61P43/00A61P5/00C07K19/00
Inventor D·安特尔曼R·J·格雷戈里K·N·维尔斯
Owner CANJI
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