A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), which contain large fragments of genomic DNA, have been successfully used as transgenes to create mouse models of dose-dependent diseases. They are also potentially valuable as transgenes for dominant diseases given that point mutations and/or small rearrangements can be accurately introduced. Here, we describe a new method to introduce small alterations in BACs, which results in the generation of point mutations with high frequency. The method involves homologous recombination between the original BAC and a shuttle vector providing the mutation. Each recombination step is monitored using positive and negative selection markers, which are the Kanamycin-resistance gene, the sacB gene and temperature-sensitive replication, all conferred by the shuttle plasmid. We have used this method to introduce four different point mutations and the insertion of the β-galactosidase gene in a BAC, which has subsequently been used for transgenic animal production.

The type I BMP receptor BmprIB is essential for female reproductive function

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.

Baseline data for public education about alcohol and highway safety in Vermont.

National Highway Traffic Safety Administration, Washington, D.C.

Estimative products on Vietnam, 1948-1975.

Accompanying CD-ROM contains full text of the 38 papers included in the printed text and 138 other declassified documents.

Reference materials for collaborative tests of air quality methods /

Mode of access: Internet.

Nomenclatura comercial.

Compilers: John K. Chandler, John C. Redman, Virginia H. Wood, Caroline Larner.

Dick Wilson,: the rumseller's victim: or, Humanity pleading for the "Maine Law". A temperance story founded on fact ...

Mode of access: Internet.

Quality assurance of chemical measurements /

Cover title.

Vocational education in public schools as related to social, economic, and technical trends.

Cooperative research project 385.

Situational factors in leadership.

"Based on the author's dissertation ... submitted to the faculty of the Graduate School of the University of Maryland."--p. vii (revised) inserted.

Memorials of the old Chicago library, formerly Young men's association, and of the advent of the new.

Mode of access: Internet.

I. Spanish spoliations, 1795. II. French indemnity, 1803. III. French indemnity, 1831.

"Notes on some of the questions decided by the Board of Commissioners under the convention with France, of 4th July 1831" [by John Kane]: p. [377]-442.

Istorii︠à politychnoĩ ekonomiĩ /

Mode of access: Internet.

Abundance of trace and minor elements in organic and mineral fractions of coal /

"August 1980."