Nathaniel Heintz

Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kγ origin for DNA replication, the E. coli RecA gene for recombination, and the SacBgene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a “built-in” resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

The anatomical complexity of the mammalian central nervous system (CNS) presents special problems for the analysis of CNS gene expression and function. The most difficult challenge is presented by the simple fact that there are hundreds of functionally and morphologically defined cell types in the CNS. Given this complexity, the interpretation of CNS phenotypes is often problematic. The preparation of transgenic mice carrying marked bacterial artificial chromosomes (BACs) provides an important avenue for improving our understanding of CNS-expressed genes and phenotypes. This approach can allow efficient analysis of patterns of gene expression, subcellular localization of their encoded products and neuronal projection patterns. BAC transgenic mice can also provide access to information relevant to gene function based on phenotypes arising from increased gene dosage or expression of activating and dominant-negative alleles. This review will concentrate on these issues and their relevance to the analysis of CNS-expressed genes.

▪ Abstract Thanks largely to cloning the genes for several neurodegenerative diseases over the past decade and the existence of mouse mutants, the molecular basis of neurodegeneration is finally beginning to yield some of its secrets. We discuss what has been learned about the pathogenesis of “triplet repeat” diseases through mouse models for spinocerebellar ataxia types 1 and 3 and Huntington disease, including the roles of nuclear aggregates and protein cleavage. We also discuss the neurologic phenotypes that arise from mutations in neurotransmitter receptors (lurcher mice) and ion channels (weaver, leaner, and tottering mice), drawing parallels between ischemic cell death and the neurodegeneration that occurs in the lurcher mouse. Finally, we discuss common mechanisms of cell death and lessons learned from these mouse models that might have broader relevance to other neurologic disorders.

Lurcher (Lc) is a spontaneous, semidominant mouse neurological mutation. Heterozygous lurcher mice (Lc/+) display ataxia due to a selective, cell-autonomous, apoptotic death of 90% of cerebellar Purkinje cells during postnatal development. Homozygous lurcher mice (Lc/Lc) die shortly after birth due to massive loss of mid- and hindbrain neurons during late embryogenesis. We identified the mutations responsible for neurodegeneration in two independent Lc alleles as identical G-to-A transitions that change a highly conserved alanine to a threonine residue in transmembrane domain III of the mouse delta 2 glutamate receptor gene (GluRE2). Lc/+ Purkinje cells displayed a very high membrane conductance and a depolarized resting potential, indicating the presence of a large, constitutive inward current. Expression of the mutant GluR delta 2Lc protein in Xenopus oocytes confirmed these results, demonstrating that lurcher is an inherited neurodegenerative disorder resulting from a gain-of-function mutation in a glutamate receptor gene. Further characterization of GluR delta 2 signaling and the activation of apoptotic death in Lc Purkinje cells have begun to yield mechanistic insights into this neurodegenerative disease, and to highlight its relationship to neuronal loss following ischemia.

The recent positional cloning and physiological characterization of the lurcher mutation resulted in the identification of a novel stimulus that results in neurodegeneration. The catastrophic loss of cerebellar Purkinje cells in lurcher heterozygotes has now been strongly associated with a large constitutive inward current which ultimately activates a programmed form of neuronal death. The completely penetrant and focal nature of the lurcher phenotype gives us an opportunity to investigate the manner in which neurons respond to an aberrant signal in the context of the brain parenchyma. Although there is no human genetic disease that is equivalent to the lurcher mutation at this time, its triggering of programmed neuronal death enables us to pose and address questions that are relevant to a large number of human neurological diseases. The advantage of working in a genetically manipulable in vivo mammalian system is evident: we can address questions relating to gene function in the nervous system in a context that is physiological. Classical genetic analyses looking for molecules that suppress or modify the lurcher phenotype are under way and have now been supplemented with two novel techniques developed in our laboratory: biolistic transfection of cerebellar slices and Bacterial Artificial Chromosome modification. The integration of these novel and classical approaches will facilitate the testing of hypotheses, developed during the course of our study of the lurcher mutation, which explore the propagation of abnormal signals and the initiation of programmed neuronal death in neurons.

Calbindin D28 encodes a calcium binding protein that is expressed in the cerebellum exclusively in Purkinje cells. We have used biolistic transfection of organotypic slices of P12 cerebellum to identify a 40-bp element from the calbindin promoter that is necessary and sufficient for Purkinje cell specific expression in this transient in situ assay. This element (PCE1) is also present in the calmodulin II promoter, which regulates expression of a second Purkinje cell Ca ²⁺ binding protein. Expression of high levels of exogenous calbindin or calretinin decreased transcription mediated by PCE1 in Purkinje cells 2.5- to 3-fold, whereas the presence of 1 μM ionomycin in the extracellular medium increased expression. These results demonstrate that PCE1 is a component of a cell-specific and Ca ²⁺ -sensitive transcriptional regulatory mechanism that may play a key role in setting the Ca ²⁺ buffering capacity of Purkinje cells.

Lurcher (Lc) is a semidominant mouse mutant that displays a characteristic ataxia in the heterozygous state beginning in the third postnatal week. This symptom results from a neurodegenerative event in the cerebellum: There is a catastrophic loss of Purkinje cells in the heterozygote animal between postnatal days 10 and 15. In an effort to identify the genetic lesion borne by Lc mice, we initiated a cloning project based on the position of the Lcmutation on mouse chromosome 6. We have extended our previous analysis of the genomic segment containing the Lc locus by isolating a set of stable and manipulable genomic clones called bacterial artificial chromosomes (BACs) that cover this region of mouse chromosome 6. These clones provided a good substrate for the isolation of markers that were used to refine the physical map of the locus. Furthermore, 20 of these markers were mapped onto our (B6CBACa-A^w − J/A − Lc × CAST/Ei)F₁ × B6CBACa-A^w − J/Abackcross, refining the genetic map and identifying two nonrecombinant markers (D6Rck354 and D6Rck355). These two markers, in conjunction with the closest flanking markers, were used to identify a 110-kb genomic segment that contains all four markers and hence contains the Lc locus. This small genomic segment, covered by multiple BACs, sets the stage for the final effort of this project—the identification of transcripts and of the mutation within theLc locus.[The Lt1 sequence has been submitted to GenBank as two ESTs; the accession numbers are U89356 andU89357.]