Identification and characterization of a subpopulation of CD133+ cancer stem-like cells derived from SK-UT-1 cells.

BACKGROUND: Uterine leiomyosarcoma (ULMS) is a malignant tumor found in the smooth muscle lining the walls of the uterus. Cancer stem cells (CSCs) are responsible for metastasis, drug resistance, and relapse of cancer, resulting in treatment failure. However, little is known about CSCs and their associated-markers in ULMS. We aimed to characterize and identify a subpopulation of CD133+ cancer stem-like cells derived from SK-UT-1 cell line. METHODS: SK-UT-1 cells were sphere-forming cultured in vitro. We also sorted the CD133+ cells derived from SK-UT-1 cell line by immunomagnetic beads. CD133+ subpopulation and apoptotic cells were detected by flow cytometry. Self-renewal and anchorage-independent growth capabilities were examined using sphere and colony formation assays. The tumorigenicity of the fourth-passage spheres and parental SK-UT-1 cells was used by mouse xenograft model in vivo. Cell proliferation ability and sensitivity to doxorubicin (DXR) were assessed by CCK-8 assay. Cell migration and invasion were tested by wound healing assay or Transwell migration and invasion assays. Expressions of CSC-related marker were analyzed by Western blotting. RESULTS: The fourth-passage spheres were defined as a CD133+ cell population, which was accompanied by increase of sphere and colony forming rate, migration and invasion abilities, as well as drug-resistant properties in vitro. Moreover, the fourth-passage spheres showed a stronger tumorigenic potential in vivo. CD133+ cell population sorted from SK-UT-1 line showed an increased ability in sphere and colony formation, proliferation, migration, invasion, resistance to apoptosis after treatment with doxorubicin (DXR) compared with CD133- cell population. The expression levels of CSCs-related markers (e.g., CD44, ALDH1,BMI1, and Nanog), were significantly elevated in CD133+ cells compared with those in CD133- cells. CONCLUSIONS: Collectively, our findings indicated that CD133 may be a significant marker for cancer stem-like cells, and it may be a potential therapeutic target for human ULMS.

"Perfect" designer chromosome V and behavior of a ring derivative.

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.

Bug mapping and fitness testing of chemically synthesized chromosome X.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

Robust orthogonal recombination system for versatile genomic elements rearrangement in yeast Saccharomyces cerevisiae.

Rearrangement of genomic DNA elements in a dynamic controlled fashion is a fundamental challenge. Site-specific DNA recombinases have been tamed as a powerful tool in genome editing. Here, we reported a DNA element rearrangement on the basis of a pairwise orthogonal recombination system which is comprised of two site-specific recombinases of Vika/vox and Cre/loxp in yeast Saccharomyces Creevisiae. Taking the advantage of the robust pairwise orthogonality, we showed that multi gene elements could be organized in a programmed way, in which rationally designed pattern of loxP and vox determined the final genotype after expressing corresponding recombinases. Finally, it was demonstrated that the pairwise orthogonal recombination system could be utilized to refine synthetic chromosome rearrangement and modification by loxP-mediated evolution, SCRaMbLE, in yeast cell carrying a completely synthesized chromosome III.

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.