Springer Science and Business Media LLC

Stem cells are unspecialized/undifferentiated cells that exist in embryos and adult tissues or can be converted from somatic differentiated cells. Use of stem cells for tissue regeneration and tissue engineering has been a cornerstone of the regenerative medicine. Stem cells are also believed to exist in cancer tissues, namely cancer stem cells (CSCs). Growing evidence suggests that CSCs are the culprit of cancer dormancy, progression and recurrence, and thus have recently received great attention. MicroRNAs (miRNAs) are a group of short, non-coding RNAs that regulate expression of a wide range of genes at a post-transcriptional manner. They are emerging as key regulators of stem cell behaviors. This mini review summarizes the basic biogenesis and mode of actions of miRNAs, recent progress and discoveries of miRNAs in cellular reprogramming, stem cell differentiation and cellular communication, as well as miRNAs in CSCs. Some potential of miRNAs in future biomedical applications and research pertaining to stem cells are briefly discussed.

We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.

A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.

Soil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N2 and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described. A modified S. meliloti gene encoding a His-tagged Hfq protein (HfqHis) was placed under the regulation of the native Hfq gene promoter (Phfqsm). The trans produced HfqHis protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains. This article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids. We illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering. Compared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.

Excitable cells in many endocrine and neuronal systems display rhythms with periodicities on the order of many minutes. To observe firing patterns that represent the output of these rhythms requires a recording technique that can monitor electrophysiological activity for several hours without affecting cell behavior. A targeted extracellular approach (also known as loose-patch) accomplishes this objective. Because low resistance seals (<20 MΩ) do not influence the cell membrane and because the normal intracellular milieu is maintained, this approach is the least invasive method for monitoring the endogenous electrical activity of single cells. In this report, we detail our use of this technique to record the firing patterns of gonadotropin-releasing hormone (GnRH) neurons in brain slices continuously for several hours.

Mô phỏng mạch máu (VM) là một cơ chế mới cung cấp máu cho khối u khác biệt với mạch máu nội mô (EV). VM có liên quan đến sự ác tính, xâm lấn, di căn và tiên lượng kém. Đến nay, vẫn chưa có phương pháp không xâm lấn nào để đánh giá VM in vivo. Siêu âm tăng cường tương phản (CEUS) đã được thực hiện để đánh giá các thông số định lượng của khối u ở chuột. Nhuộm CD31 bằng hóa mô miễn dịch - phản ứng đồng thời với Axit Periodic-Schiff đã được thực hiện để xác định VM hoặc EV trong mô khối u. Mối tương quan giữa các thông số tưới máu và mật độ VM đã được phân tích bằng kiểm định tương quan Pearson. Vào ngày thứ 15 sau khi tiêm khối u, mật độ EV và VM lần lượt là 31,15 ± 7,14 và 14,11 ± 2,99 trên trường 200×. Cường độ tối đa (IMAX) là 301,19 ± 191,56%, thời gian tăng (RT), thời gian đạt cực đại (TTP) và thời gian trung bình lưu thông (mTT) lần lượt là 17,38 ± 7,82 giây, 20,27 ± 9,61 giây và 58,09 ± 26,44 giây. Mật độ VM có tương quan tích cực với RT (r = 0,3598, P = 0,0226), TTP (r = 0,3733, P = 0,0177) và mTT (r = 0,6483, P < 0,0001), trong khi mật độ EV có tương quan tích cực với IMAX (r = 0,4519, P = 0,0034). Đường kính mạch máu của VM lớn hơn đáng kể so với EV (43,81 ± 5,88 μm so với 11,21 ± 4,13 μm). Ba thông số định lượng liên quan đến VM đã được thu được và mối quan hệ giữa CEUS và VM đã được thiết lập. Do đó, CEUS có thể cung cấp một phương pháp không xâm lấn mới để đánh giá VM in vivo.