Tissue specific expression of retinoblastoma protein

A retinal cell and tissue-specific technology, applied in the direction of retinoblastoma protein, mammalian protein, peptide/protein component, etc., can solve the problem that the exact way of blocking effect is not clear.

Inactive Publication Date: 2000-02-16
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 precise pathway of arrest is 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
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Effect test

Embodiment Embodiment I

[0080] A. Introduction to the E2F-RB Fusion

[0081] 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., Genesand 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 83:149-1158 (1995); Krek et al., Cell 83:149-1158 (1995); Krek et al., Cell 78:161-172 (1994)). B. Construction of the vector

[0082] Plasmid pCTM contains a CMV promoter, a triple adenoviral leader flanked by T7 and SP6 promoters, a multiple cloning site, and downstream of the multiple clonin...

Embodiment II

[0099] Tissue-specific expression of E2F-RB fusion A. Construction of recombinant adenovirus

[0100] In this experiment, recombinant adenoviruses containing the RB polypeptide under the control of a CMV promoter or the smooth muscle alpha-actin promoter were produced.

[0101] Isolation of the smooth muscle alpha-actin promoter (bases -670 to +5) by PCR from a genomic library, Reddy et al., "Structure of the human smooth muscle alpha-actin gene" J. Biol. Chem. 265:1683 -1687 (1990), Nakano et al., "Transcription regulatory elements and first intron 5' upstream of human smooth muscle (aortic type) α-actin encoding gene" Gene99:285-289 (1991)), And 5' Xho I and Avr II and 3' Xha I, Cla I and Hind III restriction enzyme sites were added 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 directly downstream of the smooth ...

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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 the control of cell growth (see review by Adams, P. and Kaelin, W. Seminars in Cancer Biology 6:99-108 (1995)). The RB locus is frequently inactivated in various human tumor cells. RB-negative / RB mutant cells can inhibit the growth of these cells in culture as well as the tumorigenic capacity of these cells 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 (eg, Dowdy et al., Cell 73:499-511 (1993)), but the precise route of arrest is unclear. [0003] Although E2F forms a complex with RB, complex formation is more efficient if an E2F-associated protein, DP-1, is present. E2F-1 and DP-1 form stable heterodimers that bind DNA (eg, Qin et al., Genes and Dev. 6-:953-964 (1992)). The DP-1-E2F complex acts to coordinate the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61K48/00A61P5/00A61P13/08C12N15/09A61P17/06A61P27/02A61P35/00A61P43/00C07K14/47C07K14/82C07K19/00
CPCC07K2319/00C07K14/4736C12N2799/022A61K48/00A61P13/08A61P17/06A61P27/02A61P35/00A61P43/00A61P5/00C07K19/00
Inventor D·安特尔曼R·J·格雷戈里K·N·维尔斯
Owner CANJI
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