Molecular Biology Core
Juanita Merchant, M.D., Ph.D.
Linda Samuelson, Ph.D.
Transgenic Mouse Program
The Transgenic Program takes advantage of the University of Michigan Transgenic Animal Model Core, which has a long history of effective service since its inception in 1989. This Core is an active facility with approximately 100 transgenic constructs and 35 gene targeting experiments initiated yearly. Center investigators will enjoy a discount on the normal recharge rates for the following major services: production of transgenic mice and rats; production of gene targeted ES cells; and production of ES cell-mouse chimeras. Additional support services are also available to Center investigators to increase the effectiveness of their research on genetically engineered animal models.
Consultation and Education
A strength of the Transgenic Program is the extensive consultation and training provided for all aspects of transgenic and ES cell gene-targeting research. Together Drs. Camper, Samuelson and Saunders have an aggregate 50 years of experience with transgenic research. They are available to meet with Center investigators and their laboratory personnel as needed to help with the design and execution of transgenic mouse experiments. The Director offices and the Program laboratories are centrally located to facilitate these interactions. The Program Web site (www.med.umich.edu/tamc/) is also an effective resource, providing researchers with basic information, references and protocols.
Transgenic Mice and Rats
Transgenic mice are produced by microinjection of DNA into fertilized eggs. Three transgenic mice are guaranteed for each transgene construct; an average of 9 are supplied. Mice are normally produced in the (C57BL/6 X SJL)F2 genetic background, although other genetic strains may be requested, including inbred strains such as C57BL/6, at additional cost. In addition to plasmid-based transgenes, large transgenes such as bacterial artificial chromosomes (BACs) can be used at no additional cost. The University of Michigan Core is unusual in its success with BAC transgenes and its guarantee to produce transgenic mice from BACs and other large DNA clones. The transgenic rat service is similar to the mouse service, with three transgenics guaranteed and BAC transgenes at no additional cost. The normal rat strain background is Sprague-Dawley, although other strains can be requested at additional cost.
Gene Targeting in ES Cells
This service is performed in conjunction with the investigator. The Program does the ES cell manipulations while the investigator is responsible for providing an analysis of the locus specific to their gene of interest. After consultation with the investigator to vet the targeting vector design and screen for homologous recombinants, the Program staff electroporates the targeting vector into ES cells and performs all cell culture. The particular ES line used is selected from a number of different germline-competent ES cell lines available in the Program facility, including 129/Sv lines (R1, D3, CJ7, Pat5) and a C57BL/6 line (Bruce4). The Program guarantees that 480 ES cell clones containing the targeting vector will be isolated and DNA prepared for analysis. The investigator screens these DNAs for targeting events. The Program staff then expands targeted clones, the investigator confirms the allele structure by careful Southern blot analysis, and the Program performs chromosome counts before targeted ES clones are selected for microinjection to transfer the targeted mutation to the mouse germline. Note that the Program has experience with the use of both Cre/lox and Flip/frt approaches.
Production of ES Cell Chimeric Mice
Mouse chimeras are prepared from selected targeted ES cell clones by microinjection into mouse blastocysts. Due to the inherent instability of ES clones, germline chimeras are not guaranteed. However, the Program has had extensive experience with blastocyst microinjection and produces an average of 10 male chimeras for each ES cell clone microinjected. The Program guarantees that at least 50 blastocysts will be microinjected for each targeted ES cell clone. Investigators are advised to microinject three targeted ES cell clones for an excellent chance of forming germline chimeras.
A variety of additional technologies are available to assist in rodent colony management. Mouse embryo cryopreservation and recovery is offered for permanent storage and re-establishment of unique strains. Conversion of pathogen infected mice and rats to specific pathogen free status is available to clean up mouse strains imported from dirty facilities. IVF is available to assist in the propagation of strains that do not breed well, or to facilitate rapid expansion of lines. Personnel training in microinjection, surgical techniques, and ES cell culture are provided, and the micromanipulation workstations can be used for other projects, such as microinjection of viral vectors into embryos. A variety of reagents are available to facilitate research, including plasmid vectors, DNA samples, and cell lines.
Viral Vector Program
Adenoviral services are designed to provide high transduction efficiency and transient, high level expression in a wide variety of cell lines, tissues and organisms. This is accomplished by providing high quality adenoviral services which include viral construction and transfection in packaging cells, isolation of viral plaques, plaque assay, titer, virus expansion and purification, and replication competent viral analysis. The Vector Program’s unique adenoviral system is constructed with a cre/loxP in vitro recombination system for efficient and accurate recombination of adenoviral genomes. Stock adenoviral vectors expressing various transgenes, including reporter genes, have been purified, aliquoted and stored and are available upon request. The Program also has a catalogue of adenoviral shuttle vectors containing various expression cassettes tailored towards high expression levels and gene silencing in various cell types. The Vector Program facility is equipped with an adenoviral system that outperforms commercial adenoviral systems and capable personnel to fully assist Center investigators in achieving their research goals.
Retroviral services are designed to provide high transduction efficiency and stable long term expression in a wide variety of cell types, tissues and organisms. This is accomplished by providing two lentiviral systems, HIV and FIV, as well as the retrovirus MMLV. Retroviral services include viral production on two scales, small scale (100 mls) and large scale (1000 mls), virus concentration, virus titer, and optimization of viral infection in any cell type. The Vector Program has various pro-viral plasmids containing various expression cassettes for gene expression and silencing, as well as several constructs containing bi-cistronic elements for co-expression of the investigator’s gene of interest and a reporter construct from one RNA message. The core services include a catalogue of stock viruses that have been aliquoted and stored and are available upon request.
Adeno-associated Viral Services
Adeno-associated virus (AAV) vectors are designed for stable long term expression without integration into the host genome in a variety of cell types. This is accomplished by providing two complete AAV serotypes, AAV2 and AAV5, as well as the AAV1 capsid used for psuedotyping with AAV 2 or 5 viral backbones. The Vector Program facility offers stock viruses of AAV2 and AAV5 containing reporter genes.
Plasmid DNA Construction and Preparation Services
The Vector Program offers a catalogue of non-viral DNA vectors designed to express inserted genes at high levels as well as replicate in bacterial cultures to high copy numbers. Other services include construction of custom plasmids, expansion and purification of plasmid DNA, determination of DNA concentration, sequence analysis, and storage of plasmid stocks. The core is capable of providing laboratory grade (GLP) expression plasmid DNA.
Real-Time PCR Services
The BioRad iCycler apparatus for quantitative real-time PCR is available to the Core’s members. This instrument allows investigators to quantify transcription level or genome copies using a 96-well format. This instrument is used by the core for performing replication competence assays, validation of transgene expression, and quantification of viral copies per cell. The machine is available to all Center members. Program services include instruction on machine use and data analysis.
Consultation is a service provided by the Vector Program for all Center members. The goal is to help investigators choose the best method to deliver their gene to cells, tissues or animals. We offer several viral systems and pro-viral plasmids to attain this goal. We encourage all first-time core users to meet with us to go over their research goals. We help investigators gain approval to work with viruses by the Biological Biosafety Review Committee.
Microarray Gene Chip Program
Affymetrix Gene Profiling Services
Affymetrix technology gene expression profiling services are offered on a full-service service basis, which includes initial RNA template quality analysis, probe labeling, fragmentation, hybridization, scanning and initial biostatistical analysis. The Program staff is experienced in the use of a variety of species-specific GeneChips, including human, mouse, rat, zebrafish, Caenorhabditis elegans, Drosophila melanogaster and Xenopus laevis. The Program infrastructure includes necessary large equipment required for Affymetrix GeneChip processing, including a Fluidics Station 450, three Hybridization Oven 640s, and a GeneChip Scanner 3000. The Core processed > 250 GeneChips in FY 2003 and > 400 GeneChips in FY 2004. Therefore, the Program facility is well-equipped to assist Center investigators who wish to examine the gene expression profiles of clinical specimens as well as diverse in vitro or in vivo model systems.
Agilent BioAnalyzer Services
The Agilent 2100 “Lab-On-A-Chip” is a microfluidics-based platform for the analysis of DNA, RNA, and protein. This instrument is used to provide investigators with an efficient, reliable technology for the analysis of RNA quality and quantity prior to performing array experiments. It also permits Program personnel to monitor the progress of probe labeling and fragmentation prior to hybridization for Affymetrix experiments. These assays require only small, nanogram quantities of RNA and facilitate the ability of Center investigators to utilize RNA from diverse sources (laser capture microdissections, whole tissues, cell lysates) for array experiments.
ABI 7900HT Quantitative PCR and SNP Genotyping Services
The ABI 7900HT apparatus for quantitative (real-time) RT-PCR and SNP genotyping is a new instrument introduced into the Program facility in 2003. This instrument allows investigators to validate transcript expression levels for genes of interest using Taqman-based methodologies in a 96- or 384-well format. The addition of this instrument allows Center investigators to conduct high-throughput validation of array-acquired data. The Program recently added a microfluidics card capability to the ABI7900HT. The Microfluidics card platform is inherently customizable, and allows investigators to profile the expression patterns of a particular set of genes of interest, e.g., genes identified through larger scale microarray analyses, groups of genes comprising a particular pathway of interest, genes that are linked by chromosomal location, etc. The instrument also supports SNP genotyping assays, and makes, use of the Applied Biosystems Assays-on-Demand or Assays-by-Design SNP primers and fluorogenic probes.
Biostatistical and Bioinformatics Support
The Biostatistics/Bioinformatics service offers experimental design consultations and initial data evaluations including normalization schemes (Quantile and rank-invariant normalization), expression modeling (Model based expression index using dChip, MAS 5.0, and RMA (Bioconductor), and sample comparisons (see above for cDNA spotted arrays). To facilitate the Bioinformatics aspect of array data-mining, Center investigators will be provided with a fully annotated list of the significant genes from a given experiment, with direct links to public database websites (e.g., Locus Link, PubMed, UniGene, etc).
Molecular Techniques Training Program
The major services offered by this Program include serving as an educational resource for molecular biology techniques, provide a luminometer to perform reporter gene assays, to train investigators in specialized molecular techniques. During the prior funding period, this Program was called the Analysis of Gene Expression program with education of the techniques offered being performed in Dr. Merchant’s laboratory or that were within her expertise. At the time those techniques included reporter gene assays, transcription factor purification and cloning, DNA-protein interactions and recombinant protein expression. While these methods are still available in her laboratory, the demand for many of the now commercialized techniques has been reduced. Nevertheless, general education and training of investigators in the newer methodologies is still required. Moreover, measurement of reporter gene assays using a luminometer remains in high demand. Certainly, as new molecular techniques emerge over the funding period, Dr. Merchant has in the past and will continue to incorporate these techniques into her own research and is therefore able to offer the education and training to Center investigators. The current services offered by this Molecular Core Program are:
Detailed methods of some of the methods offered by this Program are provided in Section J4. The principles of the technique will be explained so that investigators can make informed decisions on when and how to apply the technique. Expected results and potential pitfalls will be delineated. A feature of this Program that complements the Microarray Program will be reviewing applications for gene chip analysis. Often investigators are not sure of what the gene chip information will tell them and how to apply the information to their individual gene. Therefore Drs. Merchant and Bai will be available to discuss the rationale behind using Microarray analysis for their project. Perhaps more importantly, the Program staff will be available to assist the investigators is deciding how best to further test genes identified in the gene chip analysis. For example, investigators need to confirm that the genes showing induction or suppression are indeed real changes. This can be done by performing RNase protection, Northern blot analysis or quantitative RT-PCR. Moreover, investigators need to address whether the effect direct or indirect. This would possibly entail DNA-protein interaction assays of which ChIP analysis is now the most effective method to document an in vivo interaction. However, good immunoprecipitation antibodies must be available. If available reagents are not available, investigators would be directed to consider other DNA-protein interaction assays such as EMSAs or footprinting. To demonstrate functional significance, the investigator may need to design expression vectors or develop a cell knock-out strategy, e.g., RNA interference. These approaches would be more rapid and useful to perform prior using transgenic mouse approaches. Thus having staff members available to assist investigators in the analysis of gene expression is critical to the success of their experiments.
After reviewing reference material and receiving an explanation of the technique from Program staff, the investigator may provide his or her own samples to use during the demonstration or observe the performance of this technique by Dr. Merchant or Dr. Bai who currently service this Program.
Review of Microarray Project Applications
Drs. Merchant and Bai will review new project applications to the Microarray Program so as to minimize unnecessary orders of chip analysis that may not reveal useful information or more information than the investigator requires. In the past, most of the questions have revolved around whether certain comparisons at the gene expression level were amenable to a “high-throughput” analysis versus a more focused analysis of one or a few genes.
An aspect of gene expression that remains in constant demand is the analysis of reporter gene assays. Luciferase is the most common of the reporter genes due to low endogenous tissue activity and high sensitivity. Two types of luminescent assays are commonly used, luciferase and Renilla luciferase. In addition, there are luminescent substrates available for beta-galactosidase. Center investigators have access to the 96-well microtiter assay Wallac Victor3 1420 Multilabel luminometer (Perkin Elmer) available in Dr. Merchant’s lab at no additional cost. The Center will maintain the service agreement on the counter.