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Rocky Mountain Neurological Disorders Center Core


Microscopy Core

Bill Betz, Core Director

Mark Dell'Acqua Co-Director

The microscopy core has two components:

 

  1. Light Microscopy. The light microscopy facility is housed in RC-1 North East of the elevators. It houses a variety of microscopes including a Zeiss 510 confocal microscope that will be equipped with a metadetector for simultaneous detection of multiple emission wavelengths with funds provided by our NINDS grant. The facility also includes a deconvolution microscope, a total internal reflection (TIRF) microscope and a rotating disk confocal (see Light Microscopy Core Facility for full description). Individuals who want to use the light microscopy facility should contact Christine Romberg. Usage of the facility will be recorded, and access will be prioritized on usage history if demand exceeds capacity.
  2. Electron Microscopy. The electron microscopy facility is located in the first floor of RC-1 South (East of the elevators). A new a state-of-the-art TECNAI G1 12 BioTwin microscope was recently purchased with a SIG grant (Kathryn Howell, P.I.). This microscope is currently being installed, and dates and times for training sessions will be announced shortly. Our grant provides partial salary coverage for Dot Dill, the electron microscopy technician, who will assist individuals in sample preparation and use of the microscope. Individuals wishing to access the EM facility should contact Dot Dill.

 

Targeting Vector Core

The core specializes in generating gene targeting and transgenic mouse models.  The core has been serving the University of Colorado community since 2005.  In that time, we have made over forty vectors including knock-in, knock-out, conditional knock-out, traditional transgenic and BAC transgenic.  We have also developed specialized vector models for knock-in at the ROSA26 locus.  For gene targeting models, our success rate for correct ES cell targeting is 95% and for germline transmission is 100%.   The core also offers additional support services such as ES cell screening, BAC purification and mouse genotyping.  

For new project requests, please contact Wallace Chick.

         

Wendy Macklin - Director
Wallace Chick - Co-Director
Kris Williams - Core Manager
Xiaoyun Zhao - Professional Research Assistant

 

Vector Types

Conditional Knock-out  
This vector achieves conditional knock-out of your GOI by deleting a pertinent exon.  The exon is floxed and can be deleted by introducing Cre to the system.  The vector contains a Neomycin positive selection marker and a TK negative selection marker. 

CKO.jpg

Knock-in
This vector is useful for introducing a mutation to the genome.  The mutation could be a base pair change, an amino acid change or fusing the expression of your gene to a marker (ex. Cre).  The vector contains a Neomycin positive selection marker and a TK negative selection marker.

Knock-in.jpg

Knock-in at the ROSA26 Locus by RMCE 
This model is useful for either constitutive or conditional expression of your cDNA at the ROSA26 locus.  This method is fast, efficient and results in a more consistent expression of cDNA as compared to traditional transgenic models.  Your cDNA can be cloned into one of our pre-designed exchange vectors.  The exchange vector is then electroporated into ES cells which already contain the tagged ROSA26 allele.  Efficient exchange between the FRT and F3 sited results in incorporation of your cDNA into the ROSA26 locus. 
RMCE.jpg



Flex Switch
This model achieves a simultaneous deletion of an exon and activation of a marker by utilizing the Flex Switch technique described by Schnutgen et. al. FLEx switch.pdf  
Initially, the GOI functions normally and no marker is expressed.  By utilizing specific recombination sites, loxP and lox511, deletion of exon and activation of marker occurs simultaneously. The expression of the marker can be driven by the endogenous promoter or a strong exogenous promoter (ex. CAG).



 

 

BAC Transgenic
The expression of a marker is driven by the promoter of GOI.  The use of BACs has several advantages 
including the presence of regulatory elements and minimal positional effects.  The marker can be of 
several types including fluorescent tags and Cre derivatives.   

 

 

Prices

Member prices apply to member of the Center for Neuroscience
For clients oustide of the University of Colorado, add another 10% to the Non Member prices
Prices are valid for the 2015 fiscal year
                                                                   Member                 Non Member
Transgenic or RMCE Vector:                          $2,149.44                $4,031.02
KI/KO, BAC Transgenic Vector:                      $2,959.36                $5,525.15
Flex Switch Vector:                                       $3,470.78                $6,506.95
ES Cell Targeting (RMCE vectors):                  $2,967.06                $3,779.56
ES Cell Screening (per 96-well plate):             $447.85                   $832.72
BAC purification:                                           $119.12                   $311.56
Gel Purification:                                            $121.97                   $207.50
Mouse Genotyping (per sample):                    $6.77                       $11.04

Zebrafish Transgenic Core

Angie Ribera, Director

The NINDS P30 Center Zebrafish Core has three Specific Aims:

  1. Create transgenic lines of zebrafish that express different fluorescent proteins in specific populations of pre- and postsynaptic neurons
  2. Create transgenic zebrafish strains that express genetically-encoded calcium indicator dyes in specific neuronal populations
  3. Maintain transgenic and wild type zebrafish strains for the UCD neuroscience community

During the first year of the grant, the fish facility at the Fitzsimons campus is being optimized and we are focusing on Aim 3. In subsequent years, Aims 1 and 2 will be a priority. The Core can cover the expenses associated with breeding wild type fish for pilot experiments. If you would like to use zebrafish embryos for pilot experiments, please contact Angie Ribera for further information.


Zebrafish transgenic lines express GFP in specific neuronal subpopulationsZebrafish transgenic lines express GFP in specific neuronal subpopulations. In the Figure, GFP expression is driven by the flh promoter. (This transgenic line was developed by Drs. Marnie Halpern and Josh Gamse - Carnegie Institute of Washington). At 48 hours post fertilization, GFP expression (green) is present in specific populations of motor neurons that extend axons either ventrally (down, asterisks) or dorsally (up, arrowhead). A subset of the axons also expresses the zn-8 epitope (red).  Zn-8 is a marker for axons of later-born (secondary) motor neurons.
Scale Bar: 25 µm.

Machine Shop

ATTENTION USERS:
Due to the need for a temporary leave of absence, the services of the Neuroscience machine shop are suspended effectively immediately until early January 2014. My apologies for any inconvenience this may cause over the next several weeks.

Ulli Bayer, Core Director

 

The Neuroscience core machine shop is located in room NG003 on the ground floor of Building 500 (northwest corner) at the Anschutz Medical Campus (View Map). The shop's equipment and staff are there to meet the custom design and machining needs of all the faculty, staff and students of the UC Denver-Anschutz Medical Campus.

 

Click to view larger image of LAGUN 

 

The shop machinery includes:

 
    • 1 manual milling machine with digital readout
    • 1 CNC milling machine (full 3-axis automation)
    • 2 Lathes (one high-precision)
    • 3 wood/metal-cutting band saws
    • 1 table saw
    • 1 precision surface grinder
    • 1 drill press
    • 1 12" disk/6" belt sander
    • 1 52" shear
    • Extensive tooling & precision measuring devices
 

Click to view larger image of SHELDONThe shop routinely stocks a modest amount of material (acrylic, aluminum, brass, hardware) for small projects.  Shop labor is provided at no cost to members of the Neuroscience Program.The shop labor rate is $43.97/hour for members of the Neuroscience Program and $87.45 for all others. Charges are based upon project time and material costs. Payment for services is made with a current university speedtype number.

 

The shop is staffed by one machinist 24 hours/week although there are currently no routine hours of operation. Contact Michael Hall at 303-724-1335 or, preferably, by e-mail at Michael.Hall@ucdenver.edu to discuss a project, to arrange a meeting or with questions.