This chapter concentrates on the use of fluorescence in situ hybridization (FISH) for genomic investigations in the laboratory rat (Rattus norvegicus). The selection of protocols included in the ...chapter has been inspired by a comprehensive range of previously published molecular cytogenetic studies on this model organism, reporting examples of how FISH can be applied for diverse investigative purposes, varying from comparative gene mapping to studies of chromosome structure and genome evolution, to characterization of chromosomes aberrations as well as transgenic insertions. The protocols, which include techniques for the preparation of mitotic chromosomes and DNA fibers from short-term cell cultures, have been gathered through the years and repeatedly tested in our laboratory, and all together aim at providing sufficient experimental versatility to cover a broad range of cytogenetic and cytogenomic applications.
Chromosome band 10q24 is rich in genes involved in development, tumorigenesis, neurological disorders, hormone metabolism, and environmentally induced disease susceptibility. We have constructed an ...STS-based integrated physical and genetic map of 10q24 derived from the CEPH–Généthon mega-YAC contig data for this region. This map consists of 42 fluorescencein situhybridization-mapped overlapping CEPH mega-YACs spanning approximately 15 Mb to which 49 STS markers have been assigned, including 24 Généthon CA repeat genetic markers, 10 known gene loci from the 10q24 region (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B,andPAX2) and 11 additional expressed sequences of unknown function.
Specific chromosomal aberrations might indicate the position of genes responsible for a particular disease. Neuroblastoma is characterized by frequent deletions and/or rearrangements of the ...subtelomeric 1p region which, accordingly, is believed to host one or more oncosuppressor gene(s) directly or indirectly involved in the development of this and other tumors. Identification of these genes could be facilitated if cell lines with well characterized interstitial deletions or reciprocal translocations could be available for application of positional cloning strategies. In the present report we present additional and novel molecular data on three well established neuroblastoma cell lines (NLF, NMB and NGP). In one of these we have identified two sites that might be good candidates for hosting oncosuppressor genes; one of these is flanked by the D1S47 and ENO1 loci while the other is distal to the A12M2 locus.
Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem ...cell (iPSC)-derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm
) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.
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