An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

Identification of the MuRF1 skeletal muscle ubiquitylome through quantitative proteomics

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

Modeling High-grade serous ovarian carcinoma using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing

Ovarian cancer remains the most lethal gynecological cancer today. High-grade serous ovarian carcinoma (HGSC) is the most common and lethal type of ovarian cancer and is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR/Cas9 mediated genome editing. We mutated tumor suppressor genes associated with HGSC in two different combinations; Brca1, Tp53, Pten with/without Lkb1 and successfully generated HGSC, however, with different latencies and pathophysiology. By utilizing Cre lineage tracing in our system, we visualized peritoneal micrometastases in an immune-competent environment. Because our strategy is flexible in selecting mutation combinations and targeting areas, it would be useful for generating ovarian cancer mouse models.

Successful i-GONAD in Brown Norway Rats by Modification of in vivo Electroporation Conditions

Improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) was developed for in situ genome editing of the preimplantation embryos present within the oviductal lumen of mice. This method is based on intra-oviductal instillation of genome editing components and subsequent in vivo electroporation (EP) in the entire oviduct. Therefore, i-GONAD differs from the previous methods (i.e., zygote microinjection and in vitro EP) in producing genome-edited mice, which relied on ex vivo handling of preimplantation embryos and egg transfer to the recipient females. We have previously demonstrated that i-GONAD can be successfully applied to produce genome-edited rats, including albino Sprague-Dawley and albino Lewis rats (however, not pigmented Brown Norway [BN] rats). We observed that the successful i-GONAD was dependent on the mouse strain used; for example, in random-bred mice, such as ICR and C3H/He × C57BL/6, it was successful under relatively stringent electrical conditions but not in the C57BL/6 strain. Under less stringent conditions, i-GONAD was successful in the C57BL/6 strain. We speculated that this would also be true for i-GONAD using BN rats. On applying a current of >500 mA, we failed to obtain rat offspring (fetuses/newborns); however, i-GONAD under a current of 100-300 mA using NEPA21 (NEPA GENE) led to the production of genome-edited BN rats with efficiencies of 75%-100%. Similarly, i-GONAD, under a current of 150-200 mA using CUY21EDIT II (BEX Co.) led to the production of genome-edited BN rats with efficiencies of 24%-55%. These experiments suggest the importance of selecting the appropriate current value, depending on the rat strain used, when performing i-GONAD.

Definition of a Novel Plasmid-Based Gene Transfection Protocol of Mammalian Skeletal Muscles by Means of In Vivo Electroporation

We describe an original electroporation protocol for in vivo plasmid DNA transfection. The right hind limbs of C57 mice are exposed to a specifically designed train of permeabilizing electric pulses by transcutaneous application of tailored needle electrodes, immediately after the injection of pEGFP-C1 plasmid encoding GFP (Green Fluorescente Protein). The electroporated rodents show a greater GFP expression than the controls at three different time points (4, 10, and 15 days). The electroporated muscles display only mild interstitial myositis, with a significant increase in inflammatory cell infiltrates. Finally, mild gait abnormalities are registered in electroporated mice only in the first 48 h after the treatment. This protocol has proven to be highly efficient in terms of expression levels of the construct, is easy to apply since it does not require surgical exposure of the muscle and is well tolerated by the animals because it does not cause evident morphological and functional damage to the electroporated muscle.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice

Rationale: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. Methods: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. Results: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. Conclusion: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.