Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies.
Several methods have been used to measure the transduction of human hematopoietic cells with LV but there are few reports of validated techniques to determine vector copy distribution in a population from single-cell measures. In earlier studies, average number of vector copies have been measured in cell populations using Southern blot detection of vector sequences, calibrated on dilutions of genomic DNA and a housekeeping gene .11 More recently, quantitative PCR (Q-PCR) has been used to provide more precise measures and the technique can be calibrated on dilutions of a plasmid bearing both vector and genomic sequences to determine average vector copy number (VCN) in a human cell population transduced with a rHIV LV.12 Such average values does not provide any indication on the initial frequency of transduced cells in the target population nor does it estimate the numbers of integrations in individual transduced cells. Hematopoietic progenitor cells have the ability to form colonies arising from a single cell when cultured at low cell density in semi-solid medium. After a culture period of about 2 weeks in methylcellulose, individual colonies can be picked and the genomic DNA of the cells can be extracted from these colony-forming cells (CFC) with proteinase K and phenol/chloroform to determine the presence or absence of vector by PCR and agarose gels, thus, determining the frequency of transduced hematopoietic progenitor cells in the initially-infected population of cells.13 Protocols for the quantification of VCN in CFC by Q-PCR have been reported recently but not validated with experimental data.14 Here, we have developed a simplified method relying on a single-step genomic DNA extraction and a duplex Q-PCR method to quantify VCN in individual CFC. We have validated this approach experimentally. Transduced and cloned human cell lines were generated as controls and used to demonstrate the feasibility, sensitivity and reproducibility of this protocol. Measures of VCN in CFC have been used to evaluate various conditions for the transduction of hematopoietic progenitor cells with LV encoding the green fluorescence protein (GFP) reporter transgene (GFP-LV) or WASP (WASP-LV). Results show that the frequency of transduced CFC and the distribution of VCN in these cells could be augmented by repeated infection. This approach should be useful to optimize rHIV transduction protocols and to verify vector safety.
Colony Forming Unit Calculation Pdf Download
To assess the transduction of the different types of hematopoietic progenitor cells, we examined the effects of the GFP-LV and WASP-LV on the VCN values in each type of hematopoietic colony, combining all experimental conditions tested for each vector. This showed that the VCN were within a similar range in colony-forming units (CFU)-granulocyte, monocyte (GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies and in majority inferior to four copies per cell as 83% of the CFC were transduced with the GFP-LV (Figure 6a) and 96% of the CFC were transduced with the WASP-LV (Figure 6b). However, it is also remarked that both GFP and WASP LV generate higher VCN values in erythroid colonies (colony-forming unit, erythroid (CFU-E)/burst-forming unit, erythoid (BFU-E)) than in myeloid and/or mixed colonies (CFU-GEMM). The difference between VCN values in erythroid colonies versus other categories alone or combined, is statistically significant (P
The operating room (OR) of the hospital is a special unit that requires a relatively clean environment. The microbial concentration of an indoor OR extrinsically influences surgical site infection rates. The aim of this study was to use active sampling methods to assess microbial colony counts in working ORs and to determine the factors affecting air contamination in a tertiary referral medical center.
Microbiological air counts were measured using a one-stage viable impactor air sampler MAS-100 NT (Andersen sampler, Merck Inc., USA) at a flow rate of 100 L/min for 10 min (1000 L). The impactor was disinfected before and after each sampling. The collected air samples were plated onto trypticase soy agar and then incubated for 48 h at 35 C. Colonies on the dishes were Gram stained, and the positive or negative bacteria were observed under a microscope at 1000X. Bacterial counts were expressed as colony forming units per cubic meter (cfu/m3). Bacterial genera were also identified. If fungi were isolated, further culture with selective medium would also be carried out. In order to reduce the detection bias, the laboratory personnel was blinded from the information of ORs and surgical procedures.
Culture assays can be used to examine the ability of hematopoietic stem and progenitor cells to proliferate and differentiate in response to hematopoietic growth factors and to study their interactions with stromal cells of the hematopoietic microenvironment. These assays are used to measure the numbers/frequencies of progenitor cells in various tissues and purified cell preparations, identify cytokines and other compounds that promote or inhibit hematopoiesis, and to determine the effects of manipulations such as cell processing, cryopreservation, ex vivo expansion and genetic modification on the viability and functional properties of the cells. Culture assays can detect hematopoietic cells at different stages of differentiation, from HSCs to lineage-restricted progenitor cells. In the following sections, the principles and applications of two of the best characterized and quantitative culture assays, the colony-forming unit (CFU) assay and the long-term culture-initiating (LTC-IC) assay, will be discussed. 2ff7e9595c
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