Practical issues in high-speed cell sorting.

Modern flow cytometric cell sorters are all capable of so-called "high-speed sorting." However, there is confusion about exactly how fast a "high-speed" cell sorter can sort cells. There are many considerations in achieving the fastest sorting speed, as well as the highest quality sort results--cell recovery, purity, and functionality. This requires the same considerations required for "slow-speed sorting"; however, a more precise implementation is required for high-speed sorting. The modern cell sorters enable high-speed sorting because of advances in high-speed electronics and data processing. We discuss the practical considerations of high-speed sorting in terms of the theory and practical aspects of the mechanical and software components of sorting, statistics of sorting, cell preparation and viability, instrument setup, sort strategies, and biosafety.

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct.

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4(+):CD8(+) T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4(+) CD4(+) T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8(+) T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading.

Isolating gene-corrected stem cells without drug selection.

Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >/=10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a "purity" of >30%, enriched >2,000-fold and with a recovery of approximately 20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >/=3 days.

A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae.

Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.

Altering the expression in mice of genes by modifying their 3' regions.

Polymorphic differences altering expression of genes without changing their products probably underlie human quantitative traits affecting risks of serious diseases, but methods for investigating such quantitative differences in animals are limited. Accordingly, we have developed a procedure for changing the expression in mice of chosen genes over a 100-fold range while retaining their chromosomal location and transcriptional controls. To develop the procedure, we first dissected the effects in embryonic stem (ES) cells of elements within and downstream of the 3' untranslated region (UTR) of a single copy transgene at the Hprt locus. As expected, protein expression varied with the steady-state level and half-life of the mRNA. The rank order of expression with various tested 3' regions is the same in ES cells, and in cardiomyocytes and trophoblastocytes derived from them. In mice having two functionally different native genes with modified 3'UTRs, the desired expression was obtained.

Phenotype correction of Fanconi anemia group A hematopoietic stem cells using lentiviral vector.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells.