Children's Medical Center Corporation

Other

Stratagene California

Stratagene

Stratagene, La Jolla, California

Raif S Geha

Jeffrey Carl Braman

John C Bauer

Dowain A Wright

Ines M Anton

Narayanaswamy Ramesh

Miguel A De La Fuente

Donald Y Leung

John H Hartwig

Described herein is a novel gene and its product, WIP, which associates with WASP. The subject invention relates to the isolated WIP gene or cDNA and transgenic mammals that have the WIP gene disrupted in their genome. Also the subject of this invention are methods of treating conditions or diseases in which WIP and/or WASP DNA or protein is deficient and/or defective, for example, mutated or altered, such that an individual is adversely affected. Also described are methods of altering or regulating WIP and its functions in a mammal or in a cell of a mammal, for example in a lymphocyte. A further subject of this invention is an assay to identify drugs which alter the activity of WIP or expression of WIP DNA.

WIP, a WASP-associated protein

The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

Circular site-directed mutagenesis

Described herein is a novel gene and its product, WIP, which associates with WASP. The subject invention relates to the isolated WIP gene or cDNA (see FIGS. A- B); nucleic acid probes, which can be fragments of the WIP gene or WIP cDNA or full-length; nucleic acid primers, which are fragments of WIP cDNA or the WIP gene; methods of assessing cells (e.g., for diagnostic purposes) for the presence of WIP DNA, (e.g., wildtype or mutated) or for the absence or occurrence of a reduced level of WIP DNA; WIP mRNA; WIP or WIP fragments, such as those which are useful to generate antibodies which bind WIP; and antibodies which bind WIP. Also the subject of this invention are methods of treating conditions in which WIP and/or WASP DNA or protein is deficient (in quantity) and/or defective (e.g., mutated/altered) such that an individual is adversely affected (e.g., has Wiskott-Aldrich Syndrome); methods of altering or regulating WASP and its functions; and methods of altering actin content, actin polymerization or both in cells, such as human lymphoid cells (e.g., &bgr; lymphocytes). A further subject of this invention is an assay to identify drugs which alter (e.g., enhance) the activity of WIP or expression of WIP DNA.

A method of treating patients having chronic, severe allergic disorders, such as atopic dermatitis or steroid-dependent asthma, with gamma interferon is disclosed. The method involves treating patients afflicated with atopic dermatitis or steroid-dependent asthma with effective dosages of gamma interferon, which reduces the clinical severity of their disease.

Treatment of atopic disorders with gamma-interferon

Growing community of inventors

Belmont, MA, United States of America

Raif S Geha