High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes

Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNos-Cd1 drive (nos-Cas9) in An. gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly co-expressed with endogenous genes in stem and pre-meiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct co-localization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.

Imatinib Mesylate Directly Impairs Class Switch Recombination through Downregulation of AID: Its Potential Efficacy as An AID Suppressor.

Abstract 3424 Activation-induced cytidine deaminase (AID), is essential for class switch recombination(CSR) and somatic hypermutation(SHM). Deregulated expression of AID acts as a genomic mutator that contributes to various tumorigenesis through chromosomal translocation and aberrant SHM. Previously, we showed that the titer of serum immunoglobulin(Ig)G and IgA in the CML patients treated with imatinib mesylate was lower than in those with interferon-ƒ¿, whereas that of IgM was higher, implying that imatinb mesylate, the abl kinase inhibitor, impairs CSR. Here we explored the effect of imatinib mesylate on CSR both in vitro and in vivo and revealed that AID was responsible for the impairement of CSR by imatinib mesylate. CSR is induced in the mouse splenic B cells by stimulation of IL-4 and LPS. In this system, IgG1 expression of spleen cells without imatinib mesylate was □‘15%, whereas that with 10ƒÊM imatinib mesylate significantly reduced to □‘3%. The reduction was observed in dose dependent manner (Figure.1). Imatinib mesylate has been reported to affect various immunomoduratory cells including dendritic cells and T cells. Our observation elucidated that imatinib mesylate has a direct effect on B cells. Figure 1 Figure 1. Next, the expression of AID and the germline transcript of IgG1, which are required for CSR was examined by use of PCR. The expression of AID significantly decreased with imatinib mesylate, whereas that of the germline transcript of IgG1 did not change with and without 10 ƒÊM imatinib mesylate (Figure.2). Since the germline transcript of IgG1 remained unchanged, it was elucidated that downregulation of AID causes the inhibition of CSR by imatinib mesylate. The similar results were obtained in the experiments where the mice injected SRBCs (a immunogen) with imatinib mesylate showed significant reduced CSR and expression of AID in the spleen. Figure 2 Figure 2. Figure 3 Figure 3. Furthermore, we investigated whether exogenous expression of AID could rescue the inhibition of CSR by imatinib mesylate. IgG1 expression in the spleen cells without imatinib mesylate was about 36%, whereas that with 10 ƒÊM imatinib mesylate reduced to about 10.2%. When AID was exogenously expressed, IgG1 expression with 10 ƒÊM imatinib mesylate reincreased to 46.2%. It was clearly documented that the reexpression of AID could almost completely cancel the effect of imatinib mesylate on CSR. Finally, trying to understand the mechanism of downregulation of AID, the expressions of the key transcription factors, such as PAX5, E2A, E2f7 and E2f8, which bind Aicda promoter region, were examined by use of the quantitative PCR. Surprisingly all of these transcriptional factors were downregulated by imatinib mesylate. Especially the expression of E2A was dramatically reduced by imatinib mesylate. E2A is the crucial transcriptional activator for AID expression, suggesting that the remarkable downregulation of E2A by imatinib mesylate may lead to the downregulation of AID expression. Taken together, these observation lead us to the conclusion that suppression of AID via E2A is responsible for inhibition of CSR by imatinib mesylate. Our findings shed light on the etiology of hypogammaglobulinemia, an adverse effect of imatinib mesylate frequently observed in the clinical settings. The assessment and close examination of the adverse effects of the kinase inhibitors is important for the future treatment for BCR-ABL leukemias, because the treatment by multiple tyrosine kinase inhibitors like tuberculosis and HIV infection is assumed to be introduced in order to overcome the problem of drug resistance induced by the mutation such as T315I or other. It is also suggest that imatinib mesylate is the potential drug for clinical usage as AID suppressor, which deregulation of CSR and SHM with the genomic instability is observed in many B cell malignancy. Disclosures: No relevant conflicts of interest to declare.

A Targeted Deletion of a Region Upstream from the Jκ Cluster Impairs κ Chain Rearrangement In Cis in Mice and in the 103/bcl2 Cell Line

We have shown previously that a mutation of the KI-KII site immediately 5′ to Jκ1 on the mouse immunoglobulin light chain κ locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement- inducible mouse 103/bcl2-hygroR pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the κ locus on the regulation of rearrangement.