Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.

Tamoxifen activity against Plasmodium in vitro and in mice

Background Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice. Methods To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria. Results Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice. Conclusions Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

EXTH-38. ENGINEERED EXOSOMES FOR THERAPEUTIC GENE DELIVERY IN BRAIN TUMORS

The poor outcome of patients with glioblastoma (GBM) is at least partly due to the inability to deliver therapeutic agents to the tumor. We have shown that exosomes, naturally occurring nano-size extracellular vesicles, are capable of delivering antiglioma microRNAs (MiRs) to brain tumors (Lang, FM et al. Neuro Oncol, 2018;20(3):380–390). However, our studies suggested that there is significant opportunity to increase packaging efficiency and delivery specificity of exosomes. To this end, we engineered exosomes to express specific viral proteins (called eExos) in order to enhance packaging and delivery capabilities of antiglioma genes. These eExos are created by transfecting HEK 293 cells with plasmids containing viral proteins and a plasmid of the therapeutic gene. After 72 hrs, differential ultracentrifugation was used to isolate the exosomes. To test the efficacy of these novel eExos, we transfected them with a plasmid containing Cre recombinase (as the therapeutic gene), and treated U87 cells harboring a dsRed/eGFP Cre recombinase/LoxP site (U87dsR/GFP). In in vitro studies, treatment of U87dsR/GFP with a single dose of eExos resulted in 82% conversion rate of cells from red to green, compared to control exosomes (< 18% green cells). In in vivo studies, a single intratumoral injection of eExos into mice harboring 7-day old intracranial U87dsR/GFP gliomas, resulted in significant increases in green cells compared to control exosomes when tumors were harvested at day 10. Mechanistic studies employing florescent microscopy demonstrate that in contrast to natural exosomes, eExos deliver their cargo to the nucleus rather than to lysosomes, avoiding degradation of the delivered agent and facilitating expression of the plasmid. We conclude that eExos, engineered to contain specific viral proteins, are capable of packaging and delivering antiglioma genes more effectively than natural exosomes and may overcome the current inability to deliver biological therapeutic agents to brain tumors.

Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2−/−) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2–6−/− mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2−/− male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.