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Programmed cell death and Ich-3

a cell death and ich technology, applied in the field of molecular biology, to achieve the effects of delayed follicular development, delayed follicle activation, and reduced endowment of primordial oocytes

Inactive Publication Date: 2003-03-13
YUAN JUNYING +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0036] In additional embodiments, this invention provides a method of testing compounds affecting follicular development by providing a transgenic non-human animal having a disrupted Ich-3 gene, wherein the animal exhibits delayed follicle activation as characterized by a reduced endowment of primordial oocytes and abnormal follicles. One administers a compound to be tested to the transgenic animal, and determines the effect of the compound on the delayed follicular development of the animal.

Problems solved by technology

This can be attributed to their defect in production of mature IL-1.beta.

Method used

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  • Programmed cell death and Ich-3

Examples

Experimental program
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Effect test

example 1

ICH-3 is a Member of the ICE Family

[0101] To identify additional members of the ICE family, a mouse thymus cDNA library (Strategene) was screened under low stringency conditions using human Ice cDNA as a probe. Two positive clones were identified. One of them was murine Ice cDNA and the other, named Ich-3 subsequently, encodes a protein similar but not identical to murine ICE.

[0102] Cloning and Construction of Plasmids

[0103] A mouse thymus cDNA library containing 10.sub.6 plaque-forming units was screened by human Ice cDNA as a probe. Hybridization was performed under low stringency at 40.degree. C. overnight in 5.times.SSPE, 20% formamide, 10.times.Denhardt's solution, 1% SDS. Filters were washed in 1.times.SSPE, 0.5% SDS twice at room temperature, and then twice at 42.degree. C. Two Ich-3 cDNA clones were originally obtained and subdloned into pBluescript II (named m29 and mNO). Additional Ich-3 clones were also obtained from the same cDNA library by direct screening with a Ich-3 ...

example 2

Ich-3 is Expressed in Many Tissues and is Induced by LPS

[0110] To study the expression pattern of Ich-3, total RNA was isolated from different tissues of mice.

[0111] Northern Blot Analysis and RT-PCR

[0112] Total RNA from different tissues of mouse was isolated by TRIzol total RNA Isolation (GIBCO BRL). For isolation of total RNA from mice stimulated by LPS, 7-10 weeks old mice were injected with LPS at dose of 40 mg / kg body weight and 5 hrs after LPS injection, tissues were isolated for RNA preparation. The .sup.32P-labeled probe was the 3'-fragment of Ich-3 generated by PCR using two primers NO2GA and mNoR. Hybridization was performed overnight at 62.degree. C. in 1% BSA, 1 mM EDTA, 0.5 M Sodium phosphate pH 7.2, and 17.5% SDS. The blots were washed in 40 mM Sodium phosphate (pH 7.2), 1 mM EDTA, 1% SDS and 70 mM NaCl at 65.degree. C. and autoradiographed. For RT-PCR, first strand cDNA was synthesized by use of total RNA and random priming with Moloney murine leukemia virus reverse ...

example 3

Overexpression of Ich-3 Induces Apoptosis

[0122] To examine whether expression of Ich-3 may be able to induce apoptosis, the same transient expression system used for Ice and Ich-1 (Miura et al., Cell 75:653-660 (1993); Wang et al., Cell 87:739-750 (1994)) was used. The mouse Ich-3 cDNA was fused with the E. coli lacZ gene and the fused gene was placed under the control of either a chicken .beta.-actin promoter (p.beta.actM24Z) or CMV promoter (pCMVM26Z). A mutant Ich-3 was generated by site-directed mutagenesis in which the Cys residue in the conserved pentapeptide QACRG domain was converted to a Gly residue. This mutant was also fused to the lacZ gene and placed under the control of chicken .beta.-actin promoter and named p.beta.actS6Z. These expression constructs were transfected into different culture cells and their ability to induce apoptosis was tested by determining the ratio of round dead blue cells to flat live blue cells.

[0123] Cell Culture and Transfection Studies

[0124] R...

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Abstract

This invention relates to modulation of programmed cell death. It also relates to transgenic non-human animals comprising a disrupted Ich-3 gene and methods of making these animals. The Ich-3 mutant animals exhibit resistance to septic shock and defects in folliculogenesis. This invention also relates to methods of using the transgenic animals to screen for compounds to treat septic shock and defective folliculogenesis. Moreover, this invention also relates to methods of treating septic shock in normal individuals by inhibiting ICH-3.

Description

[0001] This application claims the benefit of the filing date of U.S. Provisional Application 60 / 023,937, filed Aug. 9, 1996 and is incorporated herein by reference.BACKGROUND OF THE INVENTION[0003] 1. Field of the Invention[0004] The invention is generally in the field of molecular biology as related to the control of programmed cell death. The invention also relates to transgenic non-human animals comprising a disrupted Ich-3 gene. This invention further relates to methods of making and using the transgenic animals.[0005] 2. Related Art[0006] Programmed Cell Death[0007] Apoptosis, also referred to as programmed cell death or regulated cell death, is a process by which organisms eliminate unwanted cells. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and during aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1950); Ellis et al., Dev. 112:591-603 (1991); Vaux et al., Cell 76:777-779 (1994)). Programmed cell death c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C07K14/545C12N9/64C12N15/85
CPCA01K67/0276A01K2217/05A01K2217/075A01K2227/105A01K2267/02A01K2267/03A01K2267/0337C07K14/545C12N9/6475C12N15/8509
Inventor YUAN, JUNYINGWANG, SUYUEMIURA, MASAYUKIFISHMAN, JAY A.
Owner YUAN JUNYING
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