Adeno-associated viral serotypes differentially transduce inhibitory neurons within the rat amygdala.

Recombinant adeno-associated viruses (AAV) are frequently used to make localized genetic manipulations within the rodent brain. It is accepted that the different viral serotypes possess differing affinities for particular cell types, but it is not clear how these properties affect their ability to transduce specific neuronal cell sub-types. Here, we examined ten AAV serotypes for their ability to transduce neurons within the rat basal and lateral nuclei of the amygdala (BLA) and the central nucleus of the amygdala (CeA). AAV2 based viral genomes designed to express either green fluorescent protein (GFP) from a glutamate decarboxylase (GAD65) promoter or the far-red fluorescent protein (E2-Crimson) from a phosphate-activated glutaminase (PAG) promoter were created and pseudotyped as AAV2/1, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV 2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8. These viruses were infused into the BLA and CeA at equal titers and twenty-one days later tissue within the amygdala was examined for viral transduction efficiency. These serotypes transduced neurons with similar efficiency, except for AAV4 and AAV5, which exhibited significantly less efficient neuronal transduction. Notably, AAV4 and AAV5 possess the most divergent capsid protein sequences compared to the other commonly available serotypes. We found that the Gad65-GFP virus did not exclusively express GFP within inhibitory neurons, as assessed by fluorescent in situ hybridization (FISH), but when this virus was used to transduce CeA neurons, the majority of the neurons that expressed GFP were in fact inhibitory neurons and this was likely due to the fact that this nucleus contains a very high percentage of inhibitory neurons.

Characterization of DNA fragmentation events caused by genotoxic and non-genotoxic agents.

Leukemic cells have been shown to generate several classes of DNA fragments after treatment with cytotoxic cancer chemotherapy agents. However, it is unclear which of these fragmentation events are a direct effect of DNA-damaging chemotherapy agents, and which fragmentation events are caused by downstream processes, such as apoptosis. We have performed a detailed analysis of DNA fragmentation events which occur following cytotoxic chemotherapy in four representative leukemic cell lines (HL-60, Jurkat, K562, and Molt-4). We used a DNA topoisomerase II inhibitor (etoposide), an alkylating agent (melphalan), a nucleoside analog (cytosine arabinoside), and a non-genotoxic agent (N-methylformamide) to induce cell death. We studied high molecular weight and low molecular weight DNA fragmentation events, as well as the specific cleavage of the MLL breakpoint cluster region (bcr). The DNA fragments produced at late time points were largely independent of the agents used, while those generated at earlier time points showed clear differences in terms of fragment size and relative abundance, depending on the agent used. In addition, there were clear differences between cell lines in terms of size, relative abundance, and rate at which DNA fragments were produced by treatment with the same agents. We think that this survey documents the importance of studying several different cell lines, time points, and assays before reaching conclusions about the types of DNA fragments produced during treatment with cytotoxic agents, and provides a useful framework for studying a wide range of DNA fragments produced by cytotoxic agents.

ETV6-AML1 translocation breakpoints cluster near a purine/pyrimidine repeat region in the ETV6 gene.

The t(12;21)(p13;q22) translocation, fusing the ETV6 and AML1 genes, is the most frequent chromosomal translocation associated with pediatric B-cell precursor acute lymphoblastic leukemia. Although the genomic organization of the ETV6 gene and a breakpoint cluster region (bcr) in ETV6 intron 5 has been described, mapping of AML1 breakpoints has been hampered because of the large, hitherto unknown size of AML1 intron 1. Here, we report the mapping of the AML1 gene between exons 1 and 3, cloning of ETV6-AML1 breakpoints from different patients, and localization of the AML1 breakpoints within AML1 intron 1. In contrast to the tightly clustered ETV6 breakpoints, the AML1 breakpoints were found to be dispersed throughout AML1 intron 1. Although nucleotide sequence analysis of the breakpoint junctions showed several 5/7 matches for the V(D)J consensus heptamer recognition sequence, these matches were present only on the ETV6 alleles and not on the AML1 alleles, making it unlikely that the translocations were mediated by a simple V(D)J recombination mistake. Interestingly, several breakpoints as well as a stable insertion polymorphism mapped close to a polymorphic, alternating purine-pyrimidine tract in the ETV6 gene, suggesting that this region may be prone to DNA recombination events such as insertions or translocations. Finally, the presence of an insertional polymorphism within the ETV6 bcr must be recognized to avoid incorrect genotype designation based on Southern blot analysis.