Cell and Molecular Services and Analysis Lab

Providing Cell and Molecular Services to Analyze Genotypes 

As a lab within the Cooperative Center of Excellence in Hematology (CCEH) at Fred Hutch, the Cell and Molecular Services and Analysis lab provides VNTR (Variable Number Tandem Repeat), and LAM-PCR (Linear Amplification Mediated PCR) and other molecular technology to analyze genotypes. Services were established initially to assist hematological research, but the facility also offers our services to scientists in other areas of research. We also provide products, protocols, and services for cell and molecular analyses of the Major Histocompatibility Complex (MHC).

 

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Research and Consulting Services  |  Products and Protocols  |  Contact

Research and Consulting Services

Chimerism Analysis Using Flourescent VNTR

This technology distinguishes and quantifies the relative contributions of donor and recipient cells in a transplant setting. It can also be used to distinguish and track cells from any two individuals in a chimera or complex cell mixture, either in vivo or in vitro. The method is based on Variable Number Tandem Repeat (VNTR) PCR, which distinguishes alleles of highly polymorphic microsatellite regions of mammalian DNA.

Consulting on Molecular Analysis and Model Development

This category includes the design and implementation of a variety of molecular screening methods related to the animal model.

DLA Typing Services

We amplify, sequence, and identify alleles from several DLA genes in the animal MHC complex.  These DLA alleles must be matched between donor and recipient to facilitate engraftment of transplanted cells, tissues and organs, and to minimize graft versus host disease (GVHD).  They can also play a role in immune-system-related diseases.  Typically, to ensure a reasonable probability of a successful transplant, we type the class I MHC gene, DLA-88 and the class II genes, DRB1, DQA1, and DQB1.  Other DLA genes can also be typed if desired by the user.

DNA Vector Insertion Analysis Using LAM-PCR

This technology identifies insertion sites of viral and plasmid vector DNAs within a host genome. It also tracks clones of cells based on this insertion site data. The method is described in Harkey et al, Stem Cells Dev 2007 June 16(3): 381-92.

Genomic DNA Purification and Genotype Screening

We extract genomic DNA from a variety of sources and design and carry out PCR screens for analyzing genome modifications.

We are available to discuss our services or answer your questions.

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Products and Protocols

Animal Cells and Cell Lines

Bone marrow stromal lines: Several cell lines have been developed in the Torok-Storb lab from animal bone marrow stroma. These lines have been used to support hematopoiesis in vitro, and to study the cellular interactions involved in this development.

Purified hematopoietic cell populations: A state-of the-art large-scale cell processing core, makes it possible to generate immuno-magnetically enriched populations of animal hematopoietic cells, including CD34+, CD3+ and CD14+ populations. Additional populations may become available, so please inquire.

DLA Typing Protocols and Primers

MHC Class I Typing: DLA-88 is the most polymorphic Class I locus and is the primary focus of DLA class I typing.

Typical analysis of DLA-88 includes amplification of a 1.4 kb region of chromosome 12 which contains the polymorphic exons, 2 and 3 and flanking introns of DLA-88. The PCR product is amplified with a high-fidelity DNA polymerase, cloned, and sequenced.

For descriptions of most known alleles, to date, see Kennedy et al., (2001) Animal Genetics 32:193-199; Hardt et al. (2006) Tissue Antigens 67:163-165; and Venkataraman et al. (2007) Tissue Antigens 70:53-57

  • MHC Class II Typing: The most polymorphic class II genes are DLA-DRBI, DLA-DQBI and to a lesser extent, DLA-DQAI. The former two loci are the focus of most routine DLA class II typing.
  • MHC Genotyping Database: A listing of known DLA genes and alleles, as well as a suite of tools and references for DLA typing, can be found at https://www.ebi.ac.uk/ipd/mhc/.

We can provide primers and protocols if you wish to do the DLA typing yourself.

Contact Us for More Information or to Discuss Projects

Brian Hayes

Brian Hayes

Senior Staff Scientist
Phone: 206.667.4545
Fax: 206.667.4978