RESUMEN
The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.
Asunto(s)
Ingeniería Genética/métodos , Ratas Transgénicas/crecimiento & desarrollo , Cigoto/crecimiento & desarrollo , Animales , Sistemas CRISPR-Cas , Expresión Génica , Genes Reporteros , Microinyecciones/veterinaria , Modelos Animales , Ratas , Ratas Transgénicas/genéticaRESUMEN
Approximately 15% of human couples of reproductive age have impaired fertility, and the male component accounts for about half of these cases. The etiology is usually unknown, but high correlation with the increase in obesity rates is documented. In this study, we show that diet-induced and genetically obese mice display copulatory behavior comparable to controls, but the number of females impregnated by obese males is remarkably low. Screening for changes in gene expression in the male reproductive tract showed decreased Crisp4 expression in testis and epididymis of obese mice. Lack of CRISP4 in the luminal membrane of epididymal cells indicated inadequate secretion. Consistent with CRISP4 action in acrosome reaction, sperm from mice fed a high-fat diet (HFD) had decreased fertilization capacity. CRISP4 treatment of sperm from HFD mice prior to in vitro fertilization improved fertilization rate. In leptin-deficient obese and infertile mice, leptin's effect to restore CRISP4 expression and function required gonadal hormones. Our findings indicate that the obesity-induced decline in sperm motility and fertilization capacity results in part from the disruption of epididymal CRISP4 expression and secretion.
Asunto(s)
Fertilización/genética , Infertilidad Masculina/etiología , Obesidad/complicaciones , Proteínas de Plasma Seminal/genética , Espermatozoides/fisiología , Reacción Acrosómica/genética , Animales , Epidídimo/metabolismo , Femenino , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratones Transgénicos , Obesidad/genética , Proteínas de Plasma Seminal/metabolismo , Motilidad Espermática/genética , Espermatozoides/metabolismoRESUMEN
During spermatogenesis, global nucleosome removal occurs where histones are initially replaced by transition proteins and subsequently by protamines. This chromatin reorganization is thought to facilitate the compaction of the paternal genome into the sperm head and to protect the DNA from damaging agents. Histone ubiquitination has been suggested to be important for sex chromosome inactivation during meiotic prophase and nucleosome removal at postmeiotic stages. However, the mechanisms regulating these ubiquitin-mediated processes are unknown. In this study, we investigate the role of the ubiquitin ligase RNF8 during spermatogenesis and find that RNF8-deficient mice are proficient in meiotic sex chromosome inactivation (MSCI) but deficient in global nucleosome removal. Moreover, we show that RNF8-dependent histone ubiquitination induces H4K16 acetylation, which may be an initial step in nucleosome removal. Thus, our results show that RNF8 plays an important role during spermatogenesis through histone ubiquitination, resulting in trans-histone acetylation and global nucleosome removal.
Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Espermatogénesis/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Animales , Secuencia de Bases , Histona Acetiltransferasas/metabolismo , Histonas/química , Masculino , Meiosis/genética , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Nucleosomas/ultraestructura , ARN Interferente Pequeño/genética , Espermátides/metabolismo , Espermátides/ultraestructura , Espermatogénesis/genética , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.