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###### Department of Health and Human Services

###### National Institutes of Health 

# AGENCY:

National Institutes of Health, Public Health Service, DHHS. 

# ACTION:

Notice. 

# SUMMARY:

The inventions listed below are owned by agencies of the U.S. Government and are available for licensing in the U.S. in accordance with 35 U.S.C. 207 to achieve expeditious commercialization of results of federally-funded research and development. Foreign patent applications are filed on selected inventions to extend market coverage for companies and may also be available for licensing. 

# ADDRESSES:

Licensing information and copies of the U.S. patent applications listed below may be obtained by writing to the indicated licensing contact at the Office of Technology Transfer, National Institutes of Health, 6011 Executive Boulevard, Suite 325, Rockville, Maryland 20852-3804; telephone: 301/496-7057; fax: 301/402-0220. A signed Confidential Disclosure Agreement will be required to receive copies of the patent applications. 

## Enhanced Homologous Recombination Mediated by Lambda Recombination Proteins 

                    Donald L. Court, Daiguan Yu, E-Chaing Lee, Hilary Ellis, Nancy A. Jenkins, Neal G. Copeland (NCI), DHHS Reference No. E-177-00/0 filed 14 Aug 2000, Licensing Contact: Dennis Penn; 301/496-7056 ext. 211; e-mail:                     *pennd@od.nih.gov*.                 

The present invention concerns methods to enhance homologous recombination in bacteria and eukaryotic cells using recombination proteins derived from bacteriophage lambda. It also concerns methods for promoting homologous recombination using other recombination proteins. 

Concerted use of restriction endonucleases and DNA ligases allows in vitro recombination of DNA sequences. The recombinant DNA generated by restriction and ligation may be amplified in an appropriate microorganism such as E. coli, and used for diverse purposes including gene therapy. However, the restriction-ligation approach has two practical limitations: first, DNA molecules can be precisely combined only if convenient restriction sites are available; second, because useful restriction sites often repeat in a long stretch of DNA, the size of DNA fragments that can be manipulated are limited, usually to less than about 20 kilobases. 

Homologous recombination, generally defined as an exchange of homologous segments anywhere along a length of two DNA molecules, provides an alternative method for engineering DNA. In generating recombinant DNA with homologous recombination, a microorganism such as E. coli, or a eukaryotic cell such as a yeast or vertebrate cell, is transformed with an exogenous strand of DNA. The center of the exogenous DNA contains the desired transgene, whereas each flank contains a segment of homology with the cell's DNA. The exogenous DNA is introduced into the cell with standard techniques such as electroporation or calcium phosphate-mediated transfection, and recombines into the cell's DNA, for example with the assistance of recombination-promoting proteins in the cell. 

In generating recombinant DNA by homologous recombination, it is often advantageous to work with short linear segments of DNA. For example, a mutation may be introduced into a linear segment of DNA using polymerase chain reaction (PCR) techniques. Under proper circumstances, the mutation may then be introduced into cellular DNA by homologous recombination. Such short linear DNA segments can transform yeast, but subsequent manipulation of recombinant DNA in yeast is laborious. It is generally easier to work in bacteria, but linear DNA fragments do not readily transform bacteria (due in part to degradation by bacterial exonucleases). Accordingly, recombinants are rare, require special poorly-growing strains (such as RecBCD-strains) and generally require thousands of base pairs of homology. This invention teaches an improved method of promoting homologous recombination in bacteria. 

In eukaryotic cells, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence. This invention teaches methods useful for treating human genetic diseases, the creation of transgenic animals, or modifying the germline of other organisms. 

## Amelogenin Knockout Mice and Use as Models for Tooth Disease 

                    Dr. Ashok Kulkarni et al. (NIDCR), DHHS Reference No. E-167-00/0, Licensing Contact: John Rambosek; 301/                          ( print page 69950)                        496-7056 ext. 270; e-mail:                     *rambosej@od.nih.gov.*

This technology relates to transgenic knockout mice that may serve as an animal model for dental disease. Using gene-targeting techniques, mice have been created which are disrupted for the amelogenin gene. These mice lack the amelogenin protein, which is normally expressed only in the teeth. Since these mice lack this protein, they are expected to mimic an inherited tooth disorder called “amelogenesis imperfecta (AI)”. AI is an inherited condition that is transmitted as a dominant trait and causes the enamel of the tooth to be soft and thin resulting in discoloration, disintegration and disfigurement of the teeth. The damaged teeth are also susceptible to decay. The amelogenin knockout mice display an interesting tooth phenotype. Their maxillary incisors are chalky white in color and opaque in appearance. 

These changes are associated with mild attrition of incisor tips and molar cusps. Detailed analysis of this phenotype is in progress. The amelogenin knockout mice may be used as an animal model to develop therapeutic approaches to AI. 

## Transgenic Mouse Model for Tooth Disorders Such as Dentin Dysplasia and Dentinogenesis Imperfecta 

                    Drs. Thyagarajan, Sreenath, and Kulkarni (NIDCR), DHHS Reference No. E-150-00/0, Licensing Contact: John Rambosek, Ph.D.; 301/496-7056; e-mail:                     *rambosej@od.nih.gov.*

This technology describes transgenic mice that selectively overexpress transforming growth factor beta-1 (TGF-beta1) in odontoblast and ameloblast cells of teeth. Ameloblasts mainly make enamel, whereas odontoblasts make dentin. These transgenic mice mimic dental symptoms similar to those seen in common tooth disorders such as dentin dysplasia and dentinogenesis imperfecta. Both of these human dentin defects are inherited in an autosomal dominant manner and appear to be caused by abnormal dentin production by odontoblasts and associated poor mineralization of the dentin matrix. In both diseases, teeth are discolored and fractured, causing difficulties in eating food. Experimentally, these mice display discolored and fractured teeth with defective dentin. This transgenic mice model will be valuable to advance our understanding of the molecular pathogenesis underlying dentin dysplasia and dentinogenesis imperfecta and also for developing therapeutic strategies. 

This material is available for licensing through a PHS Biological Materials License. 

Dated: November 13, 2000.

Jack Spiegel,

Director, Division of Technology Development and Transfer, Office of Technology Transfer, National Institutes of Health. 

[FR Doc. 00-29716 Filed 11-20-00; 8:45 am] 

BILLING CODE 4140-01-P