Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors.
Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5-8 time points over 6.4-19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1-8.6) off-NSAIDs and 0.6 (95% SI 0.3-1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett’s cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.
A microfabricated platform was developed for highly parallel and efficient colony picking, splitting and clone identification. A pallet array provided patterned cell colonies which mated to a second printing array composed of bridging microstructures formed by a supporting base and attached post. The posts enabled mammalian cells from colonies initially cultured on the pallet array to migrate to corresponding sites on the printing array. Separation of the arrays simultaneously split the colonies creating a patterned replica. Optimization of array elements provided transfer efficiencies greater than 90% using bridging posts of 30 µm diameter and 100 µm length and total colony numbers of 3000. Studies using five mammalian cell lines demonstrated that a variety of adherent cell types could be cultured and effectively split with printing efficiencies of 78-92%. To demonstrate the technique’s utility, clonal cell lines with siRNA knockdown of Coronin 1B were generated using the arrays and compared to a traditional FACS/Western Blotting-based approach. Identification of target clones required a destructive assay to identify cells with an absence of Coronin 1B brought about by the successful infection of interfering shRNA construct. By virtue of miniaturization and its parallel format, the platform enabled the identification and generation of 12 target clones from a starting sample of only 3900 cells and required only 5-man hours over 11 days. In contrast, the traditional method required 500,000 cells and generated only 5 target clones with 34-man hours expended over 47 days. These data support the considerable reduction in time, manpower and reagents using the miniaturized platform for clonal selection by destructive assay versus conventional approaches.
To the Editor: In reviewing the analysis of the genomic and epigenomic landscapes of de novo acute myeloid leukemia (AML) by the Cancer Genome Atlas Research Network (May 30 issue),(1) we were struck by an apparent discrepancy in the reported clonal architecture of the AML samples as compared with that reported by Ding et al.(2) (an average of one to two distinct clones identified vs. four or more). This discrepancy may reflect the limited depth of the sequencing analysis performed in the more recent study.(1) More important, the authors do not reveal which mutations occur in the founding clone, as . . .
Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clones. This has returned the bottleneck in many of these technologies to the generation of DNA clones, and standard cloning techniques often dramatically limit the throughput and success of such processes. In order to overcome this bottleneck, higher-throughput and more parallel cloning processes need to be developed which would allow rapid, inexpensive production of final clones. In addition, there is a strong need to utilize standardized elements to avoid unnecessarily remaking fragments of clones that could be used in multiple constructs.The advent of recombinational cloning helped to increase the parallel processing of DNA clones, but was still limited by the need to generate different vector backbones for each specific need. The solution to this problem emerged with the introduction of combinatorial approaches to clone construction, based on either homologous or site-specific recombination processes. In particular, the Gateway Multisite system provides all of the necessary components for a highly parallel, inexpensive, rapid, and diverse platform for clone construction in many areas of proteomic and genomic research. Here we describe our optimized system for combinatorial cloning, including improvements in cloning protocols and construct design that permit users to easily generate libraries of clones which can be combined in parallel to create an unlimited number of final constructs. The system is capable of utilizing the tens of thousands of commercially available Gateway clones already in existence, and allows easy adaptation of most DNA vectors to the system.
CD157, encoded by BST-1, has been described as a useful flow cytometric marker for the analysis of paroxysmal nocturnal hemoglobinuria (PNH) as it is a glycosylphosphatidylinositol (GPI)-linked molecule highly expressed on normal monocytes and neutrophils. We and others observed isolated CD157 signal dropouts during intended PNH analysis. We hypothesize that these negative populations occur due to an antibody failure. To investigate the reason for this finding, we compared two different anti-CD157 antibody clones for PNH analysis.
Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza.
Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Here, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of 7 years, using high-sensitivity flow cytometry and iFISH. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T- and NK-cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer, were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers usually carrying more genetic alterations, and might be a marker of an impaired immune system indirectly associated with a poorer prognosis, particularly among females.
Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA
- Proceedings of the National Academy of Sciences of the United States of America
- Published almost 2 years ago
HIV-1-infected individuals harbor a latent reservoir of infected CD4(+) T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.
Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1(R174Q) mutation who developed at the age of 42 years a T2-ALL and, 2 years after remission, an AML-M0. Both AML-M0 and T2-ALL blast populations demonstrated a loss of 1p36.32-23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood-derived CD34(+) cells 5 years prior to T2-ALL development revealed only the missense TET2(P1962T) mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2-ALL and AML-M0 clones. This result suggests that TET2(P1962T) mutation in association with germline RUNX1(R174Q) mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.