BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

• Keywords
• CRISPR-Cas9, Conditional knockout mouse, Floxed allele, Homology-directed repair, Long single-stranded DNA, Machine learning, Mouse, Oligonucleotide, Reproducibility, Transgenesis,
• MeSH
• Alleles * MeSH
• Blastocyst metabolism   MeSH
• CRISPR-Cas Systems genetics   MeSH
• Factor Analysis, Statistical MeSH
• Microinjections MeSH
• Mice, Knockout MeSH
• Methyl-CpG-Binding Protein 2 genetics   metabolism   MeSH
• CRISPR-Associated Protein 9 metabolism   MeSH
• Regression Analysis MeSH
• Reproducibility of Results MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Mecp2 protein, mouse MeSH Browser
• Methyl-CpG-Binding Protein 2 MeSH
• CRISPR-Associated Protein 9 MeSH

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is not limited to the respiratory tract as receptors, including the angiotensin-converting enzyme 2 (ACE2), are expressed across many tissues. This study employed a new conditional mouse model, Rosa26creERT2/chACE2, which expresses human ACE2 (hACE2) across multiple organs, to investigate the effects of SARS-CoV-2 infection beyond the respiratory system. This strain demonstrated susceptibility to SARS-CoV-2 infection in a dose and sex-dependent manner, showing that infected male mice exhibited more severe disease outcomes, including significant weight loss, pronounced lung pathology and dysfunction, and increased mortality, compared to females. In contrast to intratracheal infection, intranasal virus administration facilitated viral spread to the brain, thereby underscoring the nasal route's role in the pathogenesis of neurological manifestations. Intranasal infection also led to increased innate immune system activation as compared to intratracheal virus administration, even though both routes activated the adaptive immune response. This model provides a valuable tool to study SARS-CoV-2 in individual tissues or use a multisystemic approach, and it also advances possibilities for preclinical evaluation of antiviral therapies and vaccine strategies.

• Keywords
• Conditional mouse model, Infection, SARS-CoV-2, hACE2,
• MeSH
• Angiotensin-Converting Enzyme 2 * genetics   metabolism   MeSH
• COVID-19 * pathology   virology   immunology   genetics   MeSH
• Respiratory System virology   pathology   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Lung virology   pathology   MeSH
• Immunity, Innate MeSH
• SARS-CoV-2 * pathogenicity   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ACE2 protein, human MeSH Browser
• Angiotensin-Converting Enzyme 2 * MeSH

Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

• Keywords
• acetyl-CoA: α-glucosaminide N-acetyltransferase, glycosaminoglycans, heparan sulphate, knockout mouse model, mucopolysaccharidosis,
• MeSH
• Acetyltransferases deficiency   genetics   MeSH
• Behavior, Animal MeSH
• Energy Metabolism physiology   MeSH
• Gangliosides metabolism   MeSH
• Glycosaminoglycans metabolism   MeSH
• Mitochondrial Diseases etiology   pathology   MeSH
• Mucopolysaccharidosis III complications   pathology   psychology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Neuritis etiology   pathology   MeSH
• Neurodegenerative Diseases etiology   pathology   psychology   MeSH
• Neurologic Examination MeSH
• Proteostasis Deficiencies pathology   MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetyltransferases MeSH
• Gangliosides MeSH
• glucosamine acetyltransferase MeSH Browser
• Glycosaminoglycans MeSH
• Map1lc3b protein, mouse MeSH Browser
• Microtubule-Associated Proteins MeSH

Reporter gene mouse lines are routinely used for studies related to functional genomics, proteomics, cell biology or cell-based drug screenings, and represent a crucial platform for in vivo research. In the generation of knock-in reporter lines, new gene targeting methods provide several advantages over the standard transgenic techniques. First of all, specific targeting of the genome allows expression of the reporter gene under controlled conditions, whether in a specific locus in the genome or in a "safe harbor" locus. Historically, the ROSA26 locus is used for gene knock-in strategies by homologous recombination in mouse embryonic stem cells. The other preferred place for integration of the reporter transgene in the mouse genome is the endogenous promoter of a target gene. In this study, we employed TALENs to generate a reporter fusion protein expressed from its native promoter. For monitoring DNA damage response, we generated a mouse line expressing a mCitrine-tagged version of the FANCD2 protein, involved in DNA damage response and repair, and the Fanconi anemia (FA) pathway. This model could be a valuable tool for in vivo investigation of DNA damage.

• Keywords
• DNA damage, Fanconi anemia, Mouse, TALEN, mCitrine,
• MeSH
• Bacterial Proteins MeSH
• Luminescent Proteins MeSH
• Models, Animal * MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• DNA Repair MeSH
• DNA Damage MeSH
• Fanconi Anemia Complementation Group D2 Protein genetics   MeSH
• Recombinant Fusion Proteins * MeSH
• Genes, Reporter * MeSH
• Transcription Activator-Like Effector Nucleases MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• citrine protein, bacteria MeSH Browser
• Luminescent Proteins MeSH
• Fanconi Anemia Complementation Group D2 Protein MeSH
• Recombinant Fusion Proteins * MeSH
• Transcription Activator-Like Effector Nucleases MeSH

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

• Keywords
• Animal model, Apoptotic, Caspases, Deficiency, Mouse, Non-apoptoic,
• MeSH
• Apoptosis MeSH
• Caspases * metabolism   genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Knockout * MeSH
• Mice MeSH
• Inflammation enzymology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Caspases * MeSH

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation. We show that electroporation based delivery of Cre has no negative impact on embryo survival and the method can be easily combined with in vitro fertilization resulting in a significantly faster generation of desired models. Lastly, we demonstrate that Cre protein electroporation is suitable for allelic conversion in primary cells derived from conditional mouse models.

• Keywords
• Conditional, Delivery, EC, MEF, Recombination, Transgenesis, loxP,
• MeSH
• Alleles MeSH
• Electroporation MeSH
• Integrases genetics   MeSH
• Mice MeSH
• Animals MeSH
• Zygote * MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cre recombinase MeSH Browser
• Integrases MeSH

Leishmaniasis is a complex disease caused by protozoan parasites of the genus Leishmania, which are transmitted by phlebotomine sand flies. The clinical manifestations of leishmaniasis are diverse, ranging from self-healing cutaneous lesions to fatal systemic disease. Mouse models are instrumental in advancing our understanding of the immune system against infections, yet their limitations in translating findings to humans are increasingly highlighted. The success rate of translating data from mice to humans remains low, largely due to the complexity of diseases and the numerous factors that influence the disease outcomes. Therefore, for the effective translation of data from murine models of leishmaniasis, it is essential to align experimental conditions with those relevant to human infection. Factors such as parasite characteristics, vector-derived components, host status, and environmental conditions must be carefully considered and adapted to enhance the translational relevance of mouse data. These parameters are potentially modifiable and should be carefully integrated into the design and interpretation of experimental procedures in Leishmania studies. In the current paper, we review the challenges and perspective of using mouse as a model for leishmaniasis. We have particularly emphasized the non-genetic factors that influence experiments and focused on strategies to improve translational value of studies on leishmaniasis using mouse models.

• Keywords
• experimental analysis, experimental conditions, human leishmaniasis, influencing factor, mouse model, reproducibility of data, translation,
• MeSH
• Leishmania * immunology   MeSH
• Leishmaniasis * parasitology   immunology   MeSH
• Humans MeSH
• Disease Models, Animal * MeSH
• Mice MeSH
• Reproducibility of Results MeSH
• Translational Research, Biomedical * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo. METHODS: We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult. RESULTS: We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKβ as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression. Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals. CONCLUSIONS: This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.

• Keywords
• Apoptosis, Astrocytes, Autophagy, Crigler-Najjar syndrome, ER stress, Microglia, Oxidative stress, Ugt1a1,
• MeSH
• Nerve Degeneration etiology   metabolism   pathology   MeSH
• Glucuronosyltransferase deficiency   MeSH
• Disease Models, Animal MeSH
• Cerebellum pathology   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Animals, Newborn MeSH
• Hyperbilirubinemia, Neonatal complications   pathology   MeSH
• Inflammation etiology   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucuronosyltransferase MeSH
• UGT1A1 enzyme MeSH Browser

• MeSH
• Memory, Short-Term physiology   MeSH
• Mice MeSH
• Conditioning, Operant physiology   MeSH
• Memory physiology   MeSH
• Reinforcement, Psychology * MeSH
• Models, Psychological MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin overproduction which is responsible for painful skin photosensitivity. Chronic liver disease is the most severe complication of EPP, requiring liver transplantation in some patients. Data from a mouse model suggest that cytotoxic bile formation with high concentrations of bile salts and protoporphyrin may cause biliary fibrosis by damaging bile duct epithelium. In humans, cholestasis is a result of intracellular and canalicular precipitation of protoporphyrin. To limit liver damage two strategies may be considered: the first is to reduce protoporphyrin production and the second is to enhance protoporphyrin excretion. Bile salts are known to increase protoporphyrin excretion via the bile, while heme arginate is used to decrease the production of porphyrins in acute attacks of hepatic porphyrias. The Griseofulvin-induced protoporphyria mouse model has been used to study several aspects of human protoporphyria including the effects of bile salts. However, the best EPP animal model is an ethylnitrosourea-induced point mutation with fully recessive transmission, named ferrochelatase deficiency (Fech(m1Pas)). Here we investigate the effect of early ursodesoxycholic acid (UDCA) administration and heme-arginate injections on the ferrochelatase deficient EPP mouse model. In this model UDCA administration and heme-arginate injections do not improve the protoporphyric condition of Fech(m1Pas)/Fech(m1Pas) mice.

• MeSH
• Arginine administration & dosage   therapeutic use   MeSH
• Cholagogues and Choleretics administration & dosage   therapeutic use   MeSH
• Protoporphyria, Erythropoietic chemically induced   drug therapy   MeSH
• Heme administration & dosage   therapeutic use   MeSH
• Hematopoiesis drug effects   MeSH
• Injections, Intraperitoneal MeSH
• Liver drug effects   metabolism   pathology   MeSH
• Ursodeoxycholic Acid administration & dosage   therapeutic use   MeSH
• Disease Models, Animal MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arginine MeSH
• Cholagogues and Choleretics MeSH
• Heme MeSH
• heme arginate MeSH Browser
• Ursodeoxycholic Acid MeSH