Baylor College of Medicine Intellectual and Developmental Disabilities Center

URL: http://mrrc.bcm.tmc.edu/cores/confocal.html

Keywords Gene Expression; Confocal Microscopy; Zeiss 510 systems

Description

Confocal microscopy extends the capability of conventional fluorescence microscopy by enabling the investigator to visualize a thin optical plane within a sample. Out of focus portions of the sample are minimized in the image produced. Optical sections obtained confocally are superior to those obtained by mechanical sectioning, because consecutive sections remain in precise register and can be used to produce an accurate three dimensional image or model of the original sample.

Equipment

Two Zeiss 510 systems (one upright the other inverted) are available for use by members of the MRDDRC. These newer systems incorporate many refinements and advances that improve the basic functions of older confocal systems and extend their usefulness for biological research. For example, the x,y,z stages allow automated collection of high resolution montage images of samples far larger than the field of view of a particular lens Improved automation of many collection and measurement functions both simplify use and improve mechanical precision of the systems. Time lapse and bleaching capabilities allow one to use recent methods such as Fluorescence Recovery after Photobleaching to study properties of motility of molecules within membranes and Fluorescence Resonance Energy Transfer to determine the extent of separation of molecules in the nanometer range. Live cell imaging is possible through the use of several specialized chambers for cells or tissue that permit heating and perfusion of a sample. Each microscope has an independent file server and an imaging workstation with software for preparation of publication quality figures.

Use of these systems by MRDDRC investigators has facilitated discovery and elucidation of genes involved in human disorders such as Rett and SCA1; functional analyses of genes including Math1, Mecp2, Ataxin, Gfi-1, shar-pei, VAP-33A, endophilin, crumbs, hrs, CSP, atonal, syntaxin, senseless, neurexin, ROP, discs lost, skittles, gutfeeling, synaptobrevin, synaptotagmin, apterous, Dpp and protein structure/function studies of potassium channel domains.

URL: http://mrrc.bcm.tmc.edu/cores/microarray.html

Keywords Gene Expression; Microarray Expression

Description

The objectives of the Microarray Expression Core are 1) to integrate the established Baylor Microarray Facility into the Gene Expression core to provide Baylor College of Medicine Mental Retardation Developmental Disabilities Research Center (MRDDRC) investigators access to state-of-the-art quality microarray technology and services, and 2) assist MRDDRC researchers in experimental design, data management, and data analysis.

Within the past decade we have witnessed significant advancements in research that are directly associated with the output of the genome sequencing endeavor. The results of these achievements provide hope to investigators researching complex disease including MR. In MR, complex barriers to the identification of cause include not only chromosomal abnormalities (gross and submicroscopic) but alterations in one or several genes having aberrant expression profiles or even hundreds to thousands of genes with perturbed expression. This can result in a mishmash of MR gene expression profiles that is difficult to sort through presenting a challenge to researchers attempting to elucidate the cause and effect of MR.

The genome sequencing projects, however, did not just make us realize the challenges we face as researchers into the causes of MR. The explosion of data produced created a strong new field, bioinformatics. With bioinformatics we are now able to utilize in silico (computer-based) methods to deeply mine the sequence of these genomes and produce tools that will allow us to measure the “global” gene expression profile associated with a particular cell type, tissue, or animal.

URL: http://mrrc.bcm.tmc.edu/cores/neuropathology.html

Keywords Gene Expression; Neuropathology; Histology; Hybridization

Description

The objective of the Neuropathology Laboratory is to provide a facility for the members of the MRDDRC that is equipped for and experienced in the preparation and interpretation of histology, histochemistry, immunocytochemistry, in situ hybridization preparations and ultrastructural studies of the nervous system.

The Baylor MRDDRC consists of researchers working within various areas of expertise, in which human and animal tissues are investigated in order to characterize molecular, chemical and morphologic markers. The morphologic studies include the demonstration of gene products within cells, identification of specific cell types, histochemical identification of enzyme activity, ultrastructural characterization of cell organelles, confirmation of the presence of pathognomonic lesions in human and animal study subjects, and the identification of the presence or absence of structural integrity of the nervous system in transgenic and mutant animal models of human disease. The neuropathology core provides support for these necessary studies. Careful neuropathologic and immuno-localization analyses MRDD and the function of neuronal and developmental genes.

URL: http://mrrc.bcm.tmc.edu/cores/rna.html

Keywords RNA In Situ Hybridization; Gene Expression

Description

The core provides equipment and services to determine gene expression patterns in mouse tissues. Moreover, the core will collect (or assist in collecting) specimens (normal, mutant and pathologic samples), prepare frozen sections, carry out In Situ Hybridization (ISH) and document expression patterns by microscopy. In addition, and upon request, the core will produce templates and riboprobes utilized for the in situ hybridization experiment.

The high-throughput technology on which this core facility is based is one of a kind.  A description of those features that distinguish it from the standard ISH process can be found on the Baylor College of Medicine RNA In Situ Hybridization web page.

URL: http://mrrc.bcm.tmc.edu/cores/FISH.html

Keywords Genome Analysis; Fluorescence In Situ Hybridization; FISH

Description

The objective of the Fluorescence In Situ Hybridization (FISH) core is to provide high quality and cost effective state-of-the-art molecular cytogenetic analyses (as well as conventional chromosome analyses) to all MRDDRC investigators, to enhance research efforts on disorders related to mental retardation and developmental disabilities.

Services

The FISH core provides a “special skill support” enabling MRDDRC investigators to access important methodology for genome analyses.  Major services offered by the FISH core are:

1. Provide analyses for detection and confirmation of submicroscopic DNA rearrangements; including deletions, duplications, and inversions;
2. Provide genomic mapping by FISH in human and primate cell lines;
3. Provide a simultaneous analysis of all of the chromosome end structures, or telomeres, by telomere FISH.

Genetic lesions that result in MRDD can often take the form of major alterations in the genomes of patients. This core provides access to state of the art services to MRDDRC investigators for analysis of patient genomes that is not readily duplicated in individual laboratories. The core enables investigators to perform metaphase and interphase FISH studies as well as analysis of all chromosome telomeres by FISH. In addition, mapping of specific genomic clones by FISH is available. The expertise to perform typical G-banded karyotyping and to analyze chromosomes is available through this core as well. All investigators interested in studies of patients with developmental delay or mental retardation of unknown etiology require access to an analysis of the genome that is provided through the FISH core. The expertise in the core also provides the ability for investigators to carry out more sophisticated studies, including those of other species (primates, mouse) in support of studies of models for MRDD.

This core will continue to provide conventional cytogenetic analysis as well as fluorescence in situ hybridization including metaphase FISH analysis, interphase FISH analysis, FISH mapping, and telomere FISH.

URL: http://mrrc.bcm.tmc.edu/cores/genomearrays.html

Keywords Genome Analysis; Genome-Based Arrays; Microarrays; Genome Hybridization

Description

The overall objective of the newly proposed genome-based array core is to provide MRDDRC researchers with access to genome-based microarrays for use in high-resolution genomic analyses of the human genome and comparative genome hybridization (CGH).

Services

Core services will include assistance with development of microarrays for specific research purposes, as well as production of standardized arrays that allow diagnostic and research inquiries. This core will provide Center investigators with access to unprecedented resolution for the identification and characterization of constitutional chromosomal rearrangements.

URL: http://mrrc.bcm.tmc.edu/cores/m-escell.html

Keywords Mouse Genetic Engineering; Mouse Embryonic Stem Cell Technology

Description

Many of the MRDDRC projects involve generating mutations in the germ line and somatic tissues of mice by performing homologous recombination in Embryonic Stem (ES) cells. The procedures for the manipulation of ES cells and the construction of mice are capital intensive and technically demanding. There are many different types of alleles which the Core will be able to help to establish in the mouse germ line. These alleles vary in complexity from "simple" knockouts to more sophisticated alterations generated through successive manipulations of ES cells. For example, this might include the generation of chromosomal deficiencies, the generation of conditional alleles such as flanking an exon of a gene with loxP sites and targeting Cre to specific loci. All of these allele types have been generated previously by the Core and therefore this should not present any specific problem.

Services

A summary of the types of alleles which will be generated by the Core is available on the Baylor College of Medicine Mouse Embryonic Stem Cell Technology web page. The Core will be set up to assist various projects studying mental retardation with the generation of mice with targeted mutations. Having mouse models for various MRDDRC disorders is an essential step for studies aimed at understanding pathogenesis as well as for any investigations involving therapeutic intervention. Investigators will be able to receive the assistance of the Core at several phases in their experiments:

1. Construct, design, supply of vectors and genomic libraries
2. Targeting in ES cells
3. Germline transmission of targetted alleles

URL: http://mrrc.bcm.tmc.edu/cores/m-injection.html

Keywords Mouse Genetic Engineering; Mouse Injection; Microinjected DNA; Recombinant DNA; Transgenic Mice

Description

The objective of the Microinjection Core is to generate transgenic mice by embryo microinjection for MRDDRC investigators to use in their studies of mental retardation and developmental disabilities.

Recombinant DNA can be introduced into the mouse genome by microinjection of the DNA into the pronuclei of one-cell stage mouse embryos. A fraction (usually around 20%) of the subsequent newborn mice has been found to contain the microinjected DNA stably integrated in their genome. The new DNA typically integrates as a tandem head-to-tail multimer at a single site in the genome. In most cases, the DNA integrates shortly after microinjection so that it is present in the germ line and is stably transmitted to subsequent generations. The transgenic DNA is typically transcribed and translated in a tissue-specific and stage-specific pattern which is determined by regulatory sequences included in the recombinant DNA. Consequently, transgenic mice represent an experimental system in which it is possible to add new DNA to the genome of a mammalian organism and then to study the consequences and/or pattern of expression of the new genetic information.

The DNA to be microinjected can be purified after excision from standard cloning vectors, or can be larger pieces of DNA such as intact BAC clones. The BAC clones can be engineered by homologous recombination prior to microinjection. The Microinjection Service will assist MRDDRC investigators with protocols for BAC engineering and BAC DNA purification.

Transgenic mice are being used increasingly for studies of neuronal development and neuron-specific gene expression. Experiments using transgenic mice include: a) studies of cis-acting regulatory sequences that direct gene expression to specific neuronal cell types, b) studies of inappropriate synthesis of cell-surface markers that are essential for normal neuronal morphogenesis, c) studies of neuronal maturation, cell cycle control and apoptosis, and d) studies of nuclear inclusions and RNA binding proteins. Such experiments contribute to a better understanding both of normal neurological function, and of the disorders that can lead to mental retardation.

Services

The primary function of the Microinjection Service will be to generate transgenic mice by microinjection of recombinant DNA into mouse embryos. The recombinant DNA will be constructed and provided by the laboratories of the MRDDRC investigators. The core unit will purify DNA fragments or BAC clones for microinjection, isolate the mouse embryos, microinject the DNA into the mouse embryos, re-implant the mouse embryos into pseudopregnant foster mothers, and supervise the husbandry of the newborn mice. At weaning age the mice will be ear tagged for identification, then transferred to individual investigators for further characterization. The core lab will provide advice on construction of DNAs for microinjection, screening of newborn mice for integration of microinjected DNA, and characterization of gene expression in the transgenic mice.

URL: http://mrrc.bcm.tmc.edu/cores/m-neurobehavior.html

Keywords Mouse Neurobehavior; Mutant Mouse Behavioral Analyses

Description

The objectives of the Mouse Neurobehavior Core are three-fold. The first is to provide training in the use of mouse behavioral assays. The second goal is to provide access to the Neurobehavior core for MRDDRC investigators interested in determining if there are behavioral abnormalities in their mutant mice. MRDDRC investigators will have two options available to them for the behavioral analyses of their mutant mice. Investigators will be able to either test their own mice, or they will be able to utilize core services to perform the behavioral analyses for them. The third objective is to provide training in experimental design and statistical analyses that is customized for the mutant mouse behavioral analyses.

Services

The primary purpose of the MRDDRC Neurobehavior Core is to provide MRDDRC investigators with a battery of assays that will provide initial insight into the behavioral consequences of a specific mutation. In addition, the Neurobehavior Core will also provide access and training on the use of other behavioral assays that will allow an MRDDRC investigator to perform critical secondary or follow-up studies which are important to better understand the nature of any behavioral abnormality detected with the primary behavioral test battery.

URL: http://mrrc.bcm.tmc.edu/cores/m-physio.html

Keywords Mouse Physiology; Synaptic Physiology; Hippocampal Synaptic Transmission; Synaptic Plasticity; Synaptic Physiology; Electroencephalography

Description

The Mouse Physiology Core is designed to provide MRDDRC investigators with a battery of functional assays that will provide initial insight into the neurophysiologic consequences of a specific mutation. This core is considered a significant component of the MRDDRC because it will help address the most common question following the creation of a new mouse mutant: “What is wrong with my mouse?” The MRDDRC will provide its investigators access to a battery of electrophysiologic assays that  are designed to help answer this question and direct the investigator’s attention to experiments that may directly address the role of a particular gene in generating a mental retardation or developmental disability phenotype.

The objectives of the Mouse Physiology Core are two-fold. Our first goal is to provide  MRDDRC members with the resources to determine  whether their mutant mice display derangements in neuronal synaptic transmission and synaptic plasticity, using primary cultures as well as the hippocampal slice preparation in vitro, to obtain a detailed analysis of synaptic function. Our second goal is to enable MRDDRC investigators to evaluate and correlate the development of cortical excitability and brain function with behavioral activity using continuous video/EEG monitoring in behaving mutant mice. Thus the Physiology Core will assess neuronal physiology in mouse models of human mental retardation across a broad functional range – from baseline synaptic function, to short- and long-term forms of plasticity, to the behavior of the neuron when imbedded in its native circuit in vivo. These functional assessments specifically examine the basic cellular processes that are likely to be  prominently associated with MR, including long-term alterations in synaptic function and epileptic seizures.

Services

The Mouse Physiology Core is divided into two components. The Synaptic Physiology component of the Core allows investigators to determine if the basic attributes of synaptic transmission and synaptic plasticity are intact in their animal models. The analysis of synaptic properties from autaptic cultured neurons from newborn mice is a highly reliable method to screen for basic dysfunctions in synaptic transmission. Here the analysis focuses on the behavior of individual synapses. Putative malfunction in any one of many steps that underlie the complex process of synaptic transmission can be separated and quantitatively characterized. We employ neuronal cultures from hippocampus and the striatum to look not only at brain areas highly relevant for mental retardation, but also as they allow for separation of two major classes of neurons: the inhibitory GABAergic neurons and the excitatory glutamatergic neurons. The experimental approaches screen for changes in amplitudes of evoked responses, readily releasable vesicle pool sizes, spontaneous release, vesicle filling with neurotransmitters, vesicle priming and Ca2+ triggering dynamics, postsynaptic glutamate and GABA receptor density and function. In addition, quantitative morphological analysis of morphology can be applied to identify dysfunctions in synapse formation and maintenance, as well as in imbalanced dendritic outgrowth. To investigate imbalances in excitability in neuronal networks and putative effects in synaptic plasticity we further offer hippocampal slice network physiology, given the well-documented role of the hippocampus in learning and memory. The established procedures will allow the assessment of several parameters related to normal synaptic physiology, as well as several short- and long-term forms of synaptic plasticity including paired-pulse facilitation, post-tetanic potentiation, and long-term potentiation (LTP). All of these procedures utilize extracellular recording in the hippocampal slice preparation, using standard protocols already used here on an ongoing basis.

The Video/Electroencephalography component of the Core will enable MRDDRC investigators to evaluate the development of cortical excitability and brain function over prolonged periods in behaving animal models of mental retardation produced by genetic engineering techniques. Depressed excitability or abnormal patterns of brain rhythms are among the earliest objective phenotypes of genetic human mental retardation syndromes. A high incidence of epilepsy is also associated with MR, and our facility specializes in state of the art seizure detection techniques and assessment of seizure threshold. The core has pioneered the use of chronic recordings of behaving mice utilizing 10 electrode EEG arrays, permitting mapping of focal seizures and regional rhythmic activity disturbances. The ability to correlate spontaneous EEG activity with behavioral analysis by use of synchronized video/EEG monitoring is critical to the interpretation of the mutant nervous system phenotypes studied by the MRDDRC. The core also has an extensive archive of normative data to facilitate analysis and comparisons with other epileptic mouse mutant phenotypes at different developmental ages.

URL: http://mrrc.bcm.tmc.edu/cores/tissueculture.html

Keywords Tissue Culture; Fibroblasts; Cytogenetic Analysis

Description

The objective of the Tissue Culture Core is to provide cultured cell lines to MRDDRC investigators to enhance their research efforts on disorders related to mental retardation and developmental disabilities.

Growth of primary and immortalized cell lines from normal and diseased humans is crucial for studies on the molecular basis of mental retardation. Routine tissue culture represents a "special skill support" for a laboratory and the establishment of a core facility for the development, maintenance and provision of cultured cells to a number of investigators committed to research on these diseases is a cost-effective use of manpower and resources.

Services

Studies on the molecular basis of inherited disease using state-of-the-art recombinant DNA techniques entail the use of cell lines cultured from individual patients and members of their extended pedigrees. This core represents a central tissue culture facility for the establishment, maintenance and long-term storage of skin fibroblasts and lymphoblastoid cell lines for investigators involved in the MRDDRC. This facility provides the following services to the investigators:

1. Whole blood samples obtained from patients with mental retardation and/or members of their families are used to establish EBV-transformed B-lymphoblastoid cell lines, the core facility assigns a specific number to each sample, stores the cultures appropriately, and/or returns the cell lines to investigators as they request;
2. receive primary fibroblasts, somatic cell hybrids or any specific cell-type culture for growth and long-term storage as above;
   
3. prepare specified quantities of cells from the cultures from (1) and (2) above for DNA, RNA, protein or other biochemical/cytogenetic analysis as needed;
   
4. have cultures periodically checked for mycoplasma contamination;
   
5. obtain certain cell lines commonly used in molecular biological research such as HeLa cells, retrovirus vector packaging cell lines and tissue-specific cell lines to have available as needed.
   

A critical service of the Core laboratory is the maintenance of a comprehensive database representing all cell line submissions in the history of the core. These samples are an invaluable resource for future research on the etiology and pathophysiology of mental retardation. For example, the core has continued to store samples from patients with mental retardation for research in cases where the samples were originally obtained by former MRDD investigators whom have now left BCM. These investigators, as well as current MRDD investigators have chosen our facility because of the quality of the service and the expert care for these valuable cell lines.

The core also coordinates the distribution and sharing of all samples to other investigators studying various mental retardation and developmental syndromes. The database maintains the following fields for each sample submission: (1)Patient Information, (2) Patient-Disease, (3) Disease, (4) Patient-Investigator, (5) Cell line, (6) Freezer box, (7) Freezer vial, (8) Stock information, (9) Investigator, (10) Liquid Nitrogen vial.

Significant precautions are taken to maintain confidentiality of these records including password protection and standard BCM firewalls.