An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

SummaryXer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin-resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.Method summarydif-ZeoR-dif cassettes are used to replace non-essential genes in mycobacterial genome through recombineering. Spontaneous excision of the cassette is carried out under the action of the recombinase XerCD, resulting in unmarked deletions. Subsequent rounds of mutagenesis using cassettes flanked by a range of dif site variants allow construction of multiple mutants in which the different dif sites cannot recombine which each other, yielding stable genetic constructs.

A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay.

Induction of Tolerance to FVIII in Hemophilic Mice by Delivery of Apoptotic Syngeneic Fibroblasts Expressing a FVIII Transgene.

In response to treatment with factor concentrates approximately 30% of patients with severe hemophilia A develop high titer inhibitory antibodies to Factor VIII. Inhibitor formation represents a major obstacle in treating patients of hemophilia A. We are investigating ways to prevent and treat inhibitors, in the mouse model of hemophilia A, through tolerance induction by infusion of FVIII vector modified apoptotic syngeneic fibroblasts. We generated a fibroblast cell line from a tail snip of a 129Sv−FVIIIKO mouse. We then transduced these cells with a bi−cistronic foamy virus vector expressing both the full length FVIII coding sequence and the zeocin resistance gene. Production of FVIII by these modified cells was confirmed by ELISA on cell culture medium. A cell line transduced with a control vector expressing only the zeocin resistance gene was also generated. Both vector modified cell lines were maintained under selective pressure and induced to apoptose by UV irradiation just prior to infusion into 129Sv−FVIIIKO mice. Mice were treated with two infusions of UV−irradiated FVIII expressing or control fibroblasts at three different weekly cell doses: 1 x 107, 2 x 106, or 2 x 105. Ten days later they were inoculated with 4 weekly intravenous doses of 0.2 μg albumin free recombinant human FVIII (ADVATE). An additional set of control mice were not given any apoptotic cells prior to inoculation with ADVATE. Blood was collected to determine anti−FVIII inhibitor titers one week after the final ADVATE dose. The Bethesda titers in mice treated with apoptotic FVIII expressing fibroblasts, at all 3 cell doses, were 3–5 fold lower than mice that received no cells prior to intravenous FVIII challenge (p < 0.02 for all 3 cohorts, Welch’s t−test). A pair wise comparison with mice that received equivalent numbers of control apoptotic cells demonstrated significantly lower Bethesda titers in the mice given two weekly doses of 2 x106 FVIII expressing cells (436 ± 140BU versus 1230 ± 433BU, p = 0.04). In mice that received 1 x 107 and 2 x 105 apoptotic FVIII expressing cells there was a trend for the development of lower Bethesda titers when compared to mice that received an equivalent numbers of apoptotic cells modified by the control vector. Four months after treatment with ADVATE mice from the no cell group and both the 1 x 107 control and FVIII vector expressing groups were re−challenged with 4 additional weekly doses of ADVATE. T cell proliferation assays showed the highest stimulation index in mice that received no cells and the lowest index in mice that were treated with apoptotic FVIII expressing fibroblasts. Bethesda titer results from these re−challenged mice are currently pending. Our data show that anti−Factor VIII inhibitory antibody titers can be reduced by the prior infusion of apoptotic syngeneic cells stably transduced with a FVIII expressing foamy virus vector. Furthermore, the higher titers seen in mice treated with cells containing a control vector demonstrate that the effect is dependent on delivery of FVIII within the apoptotic cells. Moreover, the T cell stimulation data seen in the mice re−challenged 4 months after initial inoculation with FVIII indicate that the decrease in immune priming is due to the induction of durable tolerance. Future work will focus on elucidating the mechanisms of tolerance induction in this model and on combining apoptotic cell delivery with other immune modulators to optimize results.

A mutant of Chlamydomonas reinhardtii that cannot acclimate to low CO2 conditions has an insertion in the Hdh1 gene

Photosynthetic microorganisms must acclimate to environmental conditions, such as low CO2 environments or high light intensities, which may lead to photo-oxidative stress. In an effort to understand how photosynthetic microorganisms acclimate to these conditions, Chlamydomonas reinhardtii was transformed using the BleR cassette, selected for Zeocin resistance and screened for colonies that showed poor growth at low CO2 levels. One of the insertional mutants obtained, named slc-230, was shown to have a BleR insert in the first exon of Hdh1, a novel, single copy gene. The predicted Hdh1 gene product has similarity to bacterial haloacid dehalogenase-like proteins, a protein family that includes phosphatases and epoxide hydrolases. In addition, Hdh1 is predicted to be localised to the chloroplast or mitochondria in C. reinhardtii. It was found that a genomic copy of wild type Hdh1 can complement slc-230. Physiological studies were conducted to determine the effects of the altered expression of Hdh1 in slc-230. slc-230 grows slowly autotrophically in low CO2, exhibits a lower affinity for inorganic carbon, a decreasing photosynthetic rate over time and a lower content of chlorophylls and quenching xanthophylls than wild type cells. Some possible roles of Hdh1 in the acclimation to low CO2 conditions are discussed.