Module 1. Tissue Culture/monoclonal antibody production


A. Description

The tissue culture/monoclonal antibody module provides the following services

  • Routine maintenance of immortalized and continuous cell lines

  • Maintenance of cryopreserved stocks of cell lines

  • Establishment of primary corneal and retinal cell lines

  • Maintenance of a tumor bank of human uveal melanoma cell lines and acquisition of new uveal melanoma cell lines from collaborators

  • Cultivation of hybridomas

  • Affinity purification and concentration of monoclonal antibodies

  • Routine screening of cell lines and hybridomas for mycoplasma and endotoxin contamination

  • Production of immortalized cell lines using SV40 large T antigen, E6/E7, or telomerase gene transfer techniques

  • Generation of adenovirus vectors and insertion of various plasmids containing cDNA gene constructs (e.g., murine IFN-β cDNA) into adenovirus vectors for gene transfer experiments.

 

This module has four fundamental goals:

1) To free the individual investigator from routine tasks in maintaining cell cultures

2) To provide investigators an inexpensive, reliable source of  monoclonal antibodies

3) To provide quality control and surveillance of cell cultures for common tissue culture contaminants (i.e., mycoplasma and endotoxin)

4) To provide expertise for more sophisticated  procedures not currently available to all investigators (e.g., construction of viral vectors for gene transfer and immortalization of primary cell cultures with the E6/E7 oncogene). 

The tissue culture/hybridoma module is directed by Dr. Jerry Niederkorn. Dr. Niederkorn has over 30 years of experience in tissue culture and hybridoma techniques. He also has experience in developing immortalized primary cell cultures.

 

B. Facilities

Routine tissue culture is performed in a 380 ft2 laboratory (E7.128), which is solely dedicated to the tissue culture module. Hybridoma cultures are maintained in a separate 960 ft2 laboratory (E7.130), also used for in vitro immunological assays. One-third of this laboratory (E7.130) is dedicated solely to the hybridoma module. These laboratories have been used in the past for tissue culture and hybridoma propagation.  The tissue culture laboratory (E7.128) is equipped with: 1) two laminar flow hoods; 2) one CO2 incubator; 3) one analytical balance; 4) one refrigerated centrifuge; 4) one refrigerator/freezer; 5) one inverted compound microscope; and 6) one compound microscope. The immunology/hybridoma laboratory (E7.130) is equipped with: 1) two biohazard negative pressure hoods; 2) two CO2 incubators; 3) one inverted compound microscope; 4) one refrigerator/freezer; 5) one refrigerated tabletop centrifuge; and 6) one compound microscope.

 

C. Services Provided

a. Routine maintenance of cell cultures
Continues and primary cell cultures are routinely maintained for individual investigators. Cell cultures are monitored every eight weeks for mycoplasma and endotoxin contamination using commercial testing kits. Backup stocks of cell cultures are stored in one of six liquid nitrogen storage tanks housed in our common equipment facility (E7.238). Backup stocks are rederived every six months. The PI’s laboratory has a cryopreserved cell bank containing: 1)15 murine tumor cell lines; 2) 20 murine hybridomas; 3) 6 E6/E7 transformed corneal epithelial and endothelial cell lines; 4) 12 human uveal melanoma cell lines; and 5) 30 different normal human and murine cell lines.

b. Hybridoma propagation and monoclonal antibody purification
Monoclonal antibodies are prepared from: 1) existing hybridomas obtained from other investigators; 2) hybridomas obtained from American Type Culture Collection (ATCC, Rockville, MD); and 3) hybridomas produced de novo by in vivo immunization of rats or mice with purified proteins, or peptides provided by participating investigators in this core facility. Our hybridoma bank contains over 20 murine hybridomas. Moreover, we have extensive experience in the purification and characterization of numerous murine monoclonal antibodies for in vitro and in vivo use. We have also generated monoclonal antibodies de novo by in vivo immunization and subsequent in vitro fusion of heterologous fusion partners from Chinese hamsters and rats, and produced IgA-secreting hybridomas de novo. Depending on the quantity of monoclonal antibodies required, hybridomas are propagated in either static flat tissue culture flasks or in roller bottle cultures. Monoclonal antibodies are purified by affinity column chromatography using protein G charged columns. The affinity purified monoclonal antibodies are dialyzed against deionized water, concentrated on a vacuum concentrator, resuspended in sterile PBS, and the protein concentration determined by BCA. The purity of the monoclonal antibodies is confirmed by western blotting with commercial anti-mouse IgG or IgM, antibodies, depending on the isotype of the monoclonal antibody.

c. Primary cell cultures and immortalization of ocular cell lines
Members of our Department of Ophthalmology have extensive experience in developing primary cell cultures from human and murine ocular tissues. These include human retinal pigment epithelial cells, murine iris/ciliary body cells, human and murine corneal epithelial cells, human and murine keratocytes, and human and murine corneal endothelial cells. Ocular cell lines established from various mutant, transgenic, and knockout mice have enormous potential for a wide variety of experiments. The tissue culture/hybridoma module are a valuable resource for such initiatives.

Investigators in our department, including the PI, have extensive experience in developing immortalized ocular cell lines using cells transformed with either the E6/E7 papilloma virus oncogene, the gene encoding the SV40 large T antigen, or the gene encoding telomerase.

d. Mycoplasma and endotoxin screening
Some investigators may wish to maintain their more fastidious cell cultures within their own laboratory, but still utilize the module’s screening service for detecting mycoplasma and endotoxin contamination. In addition to regular screening of cell cultures maintained in the core facility, we offer ad hoc screening of cell cultures that are independently maintained by investigators. Commercial kits are used to detect endotoxin and mycoplasma contamination. Cells found to be contaminated with either of these agents are destroyed and replaced with frozen stocks. Valuable cell lines that are contaminated with mycoplasma and which do not have backup frozen stocks are treated with Plasmocin (InvivoGen) to purge mycoplasma. In our hands, this has been a very effective method for eliminating mycoplasma in cell cultures.

Module 2. Imaging

 

A. Description

The Imaging Module provides equipment, infra-structural support and technical assistance to vision scientists to conduct detailed, quantitative imaging of cells, tissues, proteins, and other biomolecules. This state-of-the-art imaging facility, which includes several pieces of high-end imaging hardware, has been beneficial to NEI-funded researchers on campus, and has led to novel imaging applications and numerous high quality publications.

 

The Imaging Module has two major functions:  First, to maintain a permanent Imaging Facility that is not dependent on individual research grant support, by providing funds necessary to cover service contracts and maintenance costs so that these costs do not adversely impact the specific aims of the currently funded, NEI-sponsored research projects.  Secondly, to provide a dedicated Research Technician who will help train interested investigators and perform specific research experiments using the Facility’s instrumentation. 

 

The Module will provide a range of imaging technologies, and the expertise needed to address new research questions posed by vision scientists, therefore greatly expanding and enhancing the research efforts within our community.

 

Services provided within the Imaging Facility include:

a) Histology and Immunocytochemistry
b) Light and Fluorescence Microscopy
c) Laser Scanning Confocal Microscopy
d) Multiphoton Confocal Microscopy and Time-Lapse Imaging
e) Quantitative Morphometry and Digital Image Analysis
f) Quantitative Imaging for Autoradiography, Fluorescence, and Gel Documentation

 

B. Facilities

The Imaging Facility is housed in several dedicated rooms with a total of 766 square feet of space, including a portion of the Director’s laboratory, which provides an additional 100 square feet.  Desk space for the Technical Support person is in an adjacent room (E7.228).

Laser Scanning Confocal Microscopy Lab (E7.226A, 168 sq. ft.): 

This is a dedicated room for the Leica SP2 confocal microscope. This system features three laser for visible light excitation, and three photodetectors for simultaneous detection of three separate fluorophores. A Coherent Mira/Verdi Pulsed IR Laser is coupled to the microscope to allow multiphoton imaging. To optimize signal detection during multiphoton imaging, the microscope is configured with two non-descanned detectors. The microscope also has an environmental chamber suitable for long-term maintenance of cell viability during time-lapse imaging, and a software controllable scanning stage that can be used to create wide field montages from high magnification scans.

Wide-field Microscopy Lab (E7.212a, 80 sq. ft.):
This room houses our Leica DMI 3000 microscope equipped for standard epifluorescence and transmitted light imaging. For digital imaging, a 12-bit cooled CCD camera with 1000 line resolution (Photometrics CoolSnap FX, Roper Scientific) and a color CCD camera are interfaced to a PC running Leica Advanced Fluorescence image acquisition software.

Image Analysis Lab (E7.230, 200 sq. ft.):
This lab has three, high-end, PC Workstations and two dedicated color printers.  Each PC runs the latest version of MetaMorph for quantitative morphometric analysis and 3-dimensional reconstruction of digital images obtained with the various microscope systems. Imaris software is also available for interactive 3-D volume and surface rendering. Specialized programs developed “in-house” by Dr. Petroll for visualization and analysis of 3-D confocal microcopy datasets are also installed on each computer workstation, as is the off-line version of the Leica imaging software. The suite of programs provides a full range of imaging processing, reconstruction, visualization and analysis functions. In addition, Adobe Photoshop can be used for cropping and labeling of images for publication.

Wet lab space for Histologic Processing and Immunocytochemistry (E7.232, 150 sq. ft.):

Equipment includes a Leica Kryostat CM3050S for sectioning of frozen tissue samples; a Reichert Jung Microtome for sectioning of paraffin embedded and plastic embedded tissues; a Leica M28 Dissecting microscope for processing of tissue samples; a Revco Ultralow Freezer and other conventional refrigerators and freezers for storage of tissue samples and immunologic reagents; and a Fume Hood for formaldehyde fixed tissues and histologic staining of tissue sections.

Typhoon Lab (E7.226, 168 sq. ft.):

Equipment includes a Amersham (GE) Typhoon 9400 Variable Mode Imager, interfaced to a PC running ImageQuant software.

 

C. Services Provided

a. Histology and Immunocytochemistry

This service provides common reagents for the standard processing of formaldehyde fixed tissue and paraffin embedding, sectioning and staining for routine histology. It will also provide expertise on the processing and staining of frozen and/or paraffin embedded tissue using antigen specific antibodies. For cost efficiency, these services will be provided to those investigators requiring occasional tissue processing and the reagents used including common stains, paraffin, glass slides, mounting media, secondary antibodies, etc., will be covered as a service of the Facility. Other investigators requiring more extensive services will be required to provide personnel who will be trained on the use of the equipment and techniques.

b. Light and Fluorescence Microscopy

Collection of digital images from routine histologically and immunostained tissue sections will be collected using the Leica Diaplan Epifluorescent microscope. Investigators can have images collected by the Technical Support person for occasional samples and pilot projects or provide staff to be trained on the use of this equipment.

c. Laser Scanning Confocal Microscopy

This service can more precisely co-localize two or more structures that are stained using specific sub-cellular probes or antibodies. Up to three fluorescent probes can be imaged either simultaneously or sequentially depending on the excitation and emission characteristics of the probes.  The Technical Support person and Facility Director provide the expertise for the design of experiments and the use of appropriate probes used by investigators when necessary.  Additionally, z-series of optical sections can be collected in order to determine the localization and interrelationships between structures in 3-dimensional space. Finally, the Leica SP2 has the capability to perform fluorescence resonance energy transfer (FRET) as well as fluorescence recovery after photobleaching (FRAP) to more precisely co-localize two or more proteins within a cell and characterize the location.  These are powerful techniques that provide greater confidence in localizing the proximity of proteins to known structures or other proteins. 

d.Multiphoton Confocal Microscopy and Time-Lapse Imaging

This service addresses questions requiring either deep tissue penetration or the temporal study of live cells and tissues. Multiphoton confocal microscopy provides non-invasive tissue sectioning deeper within the tissue than is available with standard conventional or visible confocal microscopic techniques, so that thicker tissues can be analyzed. Since multiphoton confocal microscopy uses infrared light for excitation, there is less cellular phototoxicity associated with imaging of live cells.  The environmental chamber used on our system allows for time-lapse multiphoton imaging to investigate the spatial organization or temporal distribution of fluorescently tagged proteins within living cells and tissues. Again, the Technical Support person and Director will be available to assist in the design of pilot studies and collection of preliminary data. More detailed investigations will be carried out by the staff of the individual investigators. Samples for time-lapse studies will be incubated in the Tissue Culture facility prior to imaging (to minimize transport time), which is just down the hall from the confocal microscopy room.

e. Quantitative Morphometry and Digital lmage Analysis
This service provides three PC workstations that can be used to analyze images from all three microscope systems (laser and multiphton confocal, in vivo confocal, and epifluorescent). Staff using the workstations are trained by the Technical Support person on using the various software programs to analyze data. Typical applications include: 1) Reconstruction/visualization of 3-D data sets, 2) measurement of cell density, 3) morphometric measurements such as cell size and shape, 4) measurement of intensity/concentration of stained structures, and 5) distance measurements in x-y-z. Additional training is available for image processing and enhancement operations.

f. Quantitative Imaging for Autoradiography, Fluorescence, and Gel Documentation
The typhoon variable mode imager handles gel sandwiches, agarose and polyacrylamide gels, membranes, microplates, and even microarrays. Red-, green- and blue-excitation wavelengths and a wide choice of emission filters enable imaging of an extensive variety of fluorophores. Automated four-color fluorescence scanning allows multiplexing of multiple targets in the same sample ensuring accuracy of analysis, increasing throughput, and saving time. Highly sensitive optics enable direct chemiluminescent imaging without intermediate exposure to films or screens. Exceptional signal-to-noise increases the number of statistically significant differences detected. Gels are scanned between glass plates, preventing drying and shrinkage and allowing further running and rescanning if required.

Module 3. Metabolomics 


A. Description

The new Metabolomics Module has replaced the earlier Protein Biochemistry Module. However, to ensure a smooth transition, the Metabolomics facility will temporarily continue to provide many of the services originally planned for the Protein Biochemistry facility.

Specifically, the Metabolomics Module will provide assistance to investigators who work with small molecules, proteins and DNA. The following services are available:

  • Evaluation of chemical composition of biological samples (currently, with a focus on lipids) using chromatographic and mass-spectrometric approaches
  • Assistance in subcloning of cDNAs into expression vectors
  • Expression and purification of recombinant proteins in either bacterial or insect cell (baculovirus) expression systems. The facility offers the basic equipment, expertise and labor assistance necessary for all of these approaches. The requests will be dealt with in the order they are received.

 

This module has two fundamental goals:

  1. To facilitate the individual NEI investigator’s eye-related research by providing assistance in making custom biological reagents that require extensive experience, training, infrastructure and resources to be generated (such as recombinant proteins).
  2. To provide technical assistance in characterization of small molecules (such as lipids) in complex biological samples.

    Fulfilling these goals will accelerate the pace and effectiveness of NEI-funded vision research projects at UT Southwestern.

 

B. Facilities

The Metabolomics Facility is located in rooms E7.212 and E7.504.  Desk space for the Technical Support person is in room E7.504.

The module is equipped with cell incubators and shakers, a PCR instrument, an ACTA FPLC system for protein purification, a freeze-dryer, table-top centrifuges, microscopes and other small equipment. An HPLC system (Waters), an ion trap mass spectrometer and a GC/MS system (both from Thermo Electron) in Dr. Butovich’s laboratory are available for Module personnel.

Our future plans include creating a dedicated HPLC/MS system suitable for analyzing small molecules and proteins.


C. Equipment

The module is equipped with cell incubators and shakers, a PCR instrument, an ACTA FPLC system for protein purification, a freeze-dryer, table-top centrifuges, microscopes and other small equipment. An HPLC system (Waters), an ion trap mass spectrometer and a GC/MS system (both from Thermo Electron) in Dr. Butovich’s laboratory are available for Module personnel.

Our future plans include creating a dedicated HPLC/MS system suitable for analyzing small molecules and proteins.

 

D. Services Provided

Small Molecules Characterization and Quantitation

Capitalizing on a two-decade long experience of the lab members with lipids and other small molecules, the Metabolomics facility offers services related to the evaluation of the composition of complex biological samples such as cells, skin and ocular tissue extracts. The samples can be analyzed using a range of approaches which include, but are not limited to, thin layer chromatography (TLC), gas chromatography (GC), high pressure liquid chromatography (HPLC), and various types of mass spectrometry (MS). Other approaches can be considered as well.

The samples are to be provided by participating researchers in the form of extracts dissolved in proper organic solvents. The required amounts will depend on the putative composition and complexity of the mixtures, and stability of the analytes. The Module staff will assist the researchers in devising proper strategies to prepare samples suitable for analyses.

Step 1
The the samples will be evaluated to identify individual components in the mixtures, and to estimate their ranges. This is routinely done by using the direct infusion (DI) approach (no chromatographic step involved). During the DI step, the presence of interfering factors (such as exogenous contaminations) will be evaluated.

Step 2
Depending on the results of the DI analyses, a proper chromatographic technique will be chosen to 1) assure adequate separation of analytes from interfering compounds, and 2) optimize conditions of the analytical runs;

Step 3
Unknown analytes will be structurally analyzed using MS(n) approaches and compared with authentic standards (if available);

Step 4
Compounds will be quantitated using HPLC/MS (if proper standards of the analytes are available).

Initially, analyses will be offered for lipids and lipid-signaling factors. In the future, we plan on expanding our services to include other types of low-molecular weight compounds and proteins.

 

Full-length Gene Cloning Service

Our full-length cDNA cloning service offers PCR-generated clones from original cDNA libraries or cDNA generated from reverse transcription of mRNAa from special tissues or cells. We can subclone a target cDNA from an experimenter-supplied physical clone or design specific primers for the intended cDNA using its gene accession number and existing cDNAs. As part of this service, antibiotic-resistant plasmids will be transformed into E. coli and large quantities of DNA will be purified using standard commercial affinity-column purification (e.g. Qiagen Maxi-Preps). All PCR-generated clones will be fully sequenced and matched to available public records to avoid artificial mutations. We provide extensive cloning expertise for the subcloning of these cDNA into our various expression vectors, or into custom vectors (pcDNA3, etc.) needed for NEI-supported research studies.

 

Protein Expression Services

Bacterial expression of recombinant proteins: This service is divided into two phases: 1) pilot expression in small-scale E. coli (BL21 or DH5a) cultures and 2) large-scale expression of recombinant proteins for purification. If the investigators wish to construct the expression plasmids themselves, the Core staff can provide advice on plasmid construction. Once expression plasmids are generated or provided, they will be transformed into chemically-competent bacteria and small-scale cultures will be tested for the protein expression kinetics and solubility. Once optimal conditions have been determined, large-scale protein preps (up to one liter) will be performed and cells harvested at an appropriate time point.

Baculovirus/insect cell expression of recombinant fusion proteins: The baculovirus/insect cell expression system is widely used to produce recombinant proteins for many biomedical research purposes. It provides researchers with the means to generate milligram quantities of recombinant proteins with proper folding, disulfide bond formation, oligomerization and posttranslational modifications such as glycosylation, phosphorylation, acetylation, methylation, etc. In addition, the overexpressed proteins in insect cells are often localized to the same subcellular compartments as they would be in a mammalian cell. This service will also involve the pilot and large-scale expression of proteins from baculovirus-infected SF9 insect cells in culture. In short, the Bac-to-Bac Baculovirus expression system (Invitrogen) will be used by the Core to produce the recombinant proteins in cases where proper posttranslational modifications or expression of large, or enzymatically active, proteins are required. The cDNA of interest are cloned into pFastBac by the Core staff, or by P30 Core investigators themselves. The biochemistry core will then carry out the recombination process in DH10Bac cells, select and verify recombinant Bacmids, and amplify and titer the baculovirus for infection of large-scale SF9 cell cultures for recombinant protein expression. Initially, small-scale cultures of SF9 insect cells will then be infected to optimize expression conditions. If recombinant expression is successful, then larger insect cell cultures will be used for generating large-scale expression of intracellular or secreted recombinant proteins.

 

Affinity purification of expressed recombinant proteins

This service involves the solubilization and affinity purification of expressed recombinant fusion proteins by column chromatography. Using standard approaches, the bacterial or insect cell pellets will be lysed with appropriate buffers compatible with the relevant affinity resin.  The standard affinity approaches used by the facility include glutathione S-transferase (GST), six histidine (His6), and Fc fusion proteins. Commercial resins, including Ni-NTA agarose (Novagen), glutathione agarose, and protein A agarose will be used in accordance with standard protocols. The purifications will be performed on an ACTA FPLC system that allows for loading, washing, eluting, and collecting eluted protein fractions. This system also allows for additional steps of purification beyond the affinity resins using traditional column chromatography approaches (e.g. strong or weak cation or anion exchange resins, etc).