Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (␣2-␦-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the ␣2-␦-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.

Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that plays important roles during early development. The mouse egg zona pellucida is constructed of three glycoproteins, called mZPI, mZP2, and mZP3. The gene encoding mZP3 is expressed only by growing oocytes during a 2-to 3-week period of oogenesis. Here, the mZP3 gene was disrupted by targeted mutagenesis using homologous recombination in mouse embryonic stem cells. Viable female mice homozygous for the mutated mZP3 allele (mZP3-/-) were obtained. These mice are indistinguishable in appearance from wild-type (mZP3+/+) and heterozygous

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ABSTRACTpGEM-7Zf(+) was digested with Apa I and EcoRI, the recessed ends were filled-in by using T4 DNA polymerase, and the plasmid was religated (needed to destroy the Apa I site). This plasmid was digested with BamHI and ligated to a 3.8-kb BamHI fragment of mZP3 (nt 6140-10,048), generating plasmid pGEM-7*/ZP3BHI. An 800-nt PCR fragment was generated by using 1 ng of pGEM-7*/ZP3BHI as the template and the following oligonucleotides: mZP3-5'-deletion, 5'-CCCCTGCAGCGACGTCTGGTCCAAGCT-AGTTTCTCG-3'; mZP3-3'-deletion, 5 '-ATCAGGGGC-CCTACAGTGACA-3'. PCR was carried out in a 25-,ul

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