Xiangdong W. Yang

Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromsome

Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAG containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAG, and germline transmission of the intact BAG has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.

BAC-mediated gene-dosage analysis reveals a role for Zipro1 ( Ru49 / Zfp38 ) in progenitor cell proliferation in cerebellum and skin

Genetic analysis in mice has most commonly employed two general strategies: phenotypic screens for spontaneous or induced mutations and genotypic analysis using homologous recombination or gene trapping to produce deletion or insertion mutants. Here we use bacterial artificial chromosome (BAC)-mediated gene-dosage analysis in transgenic mice to reveal novel genetic functions that are not evident from conventional loss-of-function mutations. We demonstrate a role for the zinc-finger transcription factor Zipro1 (formerly Ru49 and Zfp38) in the proliferation of granule cell precursors in the developing cerebellum, and document the contribution of this process to the final stages of cerebellar morphogenesis. We also show that Zipro1 is expressed in skin, and increased Zipro1 dosage results in a hair-loss phenotype associated with increased epithelial cell proliferation and abnormal hair follicle development.

Progressive synaptic pathology of motor cortical neurons in a BAC transgenic mouse model of Huntington's disease.

Huntingtons disease (HD) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in huntingtin. A newly developed bacterial artificial chromosome transgenic mouse model (BACHD) reproduces phenotypic features of HD including predominantly neuropil-associated protein aggregation and progressive motor dysfunction with selective neurodegenerative pathology. Motor dysfunction has been shown to precede neuropathology in BACHD mice. We therefore investigated the progression of synaptic pathology in pyramidal cells and interneurons of the superficial motor cortex of BACHD mice. Whole-cell patch clamp recordings were performed on layer 2/3 primary motor cortical pyramidal cells and parvalbumin interneurons from BACHD mice at 3 months, when the mice begin to demonstrate mild motor dysfunction, and at 6 months, when the motor dysfunction is more severe. Changes in synaptic variances were detectable at 3 months, and at 6 months BACHD mice display progressive synaptic pathology in the form of reduced cortical excitation and loss of inhibition onto pyramidal cells. These results suggest that progressive alterations of the superficial cortical circuitry may contribute to the decline of motor function in BACHD mice. The synaptic pathology occurs prior to neuronal degeneration and may therefore prove useful as a target for future therapeutic design.

A rapid method for targeted modification and screening of recombinant bacterial artificial chromosome

Modification of bacterial artificial chromosomes (BACs) has been a useful method to produce genomic DNA fragments for studying gene expression and function in vitro and in vivo. The original technique involved restrictions for BAC modification and required multiple cloning steps to target sequences into the shuttle vector. Selection and screening of BAC recombinants was accomplished by drug resistance and Southern blotting. We have developed a PCR-based method for producing the modified shuttle vectors and for screening for BACs carrying homologous integrants. The combination of these techniques allows for rapid and easy targeted BAC sequence deletion or insertion.