RESUMEN
Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Fertilización In Vitro , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Infertilidad Masculina/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Oocitos/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.
Asunto(s)
Fertilidad/genética , Infertilidad Masculina/genética , Proteínas de la Membrana/genética , Familia de Multigenes/genética , Motilidad Espermática/genética , Animales , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Trompas Uterinas/fisiología , Femenino , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Espermatozoides/citología , Espermatozoides/fisiologíaRESUMEN
The N6-methylation of internal adenosines (m6A) in mRNA has been quantified and localized throughout the transcriptome. However, the physiological significance of m6A in most highly methylated mRNAs is unknown. It was demonstrated previously that the circadian clock, based on transcription-translation negative feedback loops, is sensitive to the general inhibition of m6A. Here, we show that the Casein Kinase 1 Delta mRNA (Ck1δ), coding for a critical kinase in the control of circadian rhythms, cellular growth, and survival, is negatively regulated by m6A. Inhibition of Ck1δ mRNA methylation leads to increased translation of two alternatively spliced CK1δ isoforms, CK1δ1 and CK1δ2, uncharacterized until now. The expression ratio between these isoforms is tissue-specific, CK1δ1 and CK1δ2 have different kinase activities, and they cooperate in the phosphorylation of the circadian clock protein PER2. While CK1δ1 accelerates the circadian clock by promoting the decay of PER2 proteins, CK1δ2 slows it down by stabilizing PER2 via increased phosphorylation at a key residue on PER2 protein. These observations challenge the previously established model of PER2 phosphorylation and, given the multiple functions and targets of CK1δ, the existence of two isoforms calls for a re-evaluation of past research when CK1δ1 and CK1δ2 were simply CK1δ.
Asunto(s)
Quinasa Idelta de la Caseína/genética , Relojes Circadianos/genética , Metilación , Metiltransferasas/genética , ARN Mensajero/genética , Animales , Quinasa Idelta de la Caseína/metabolismo , Masculino , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Empalme del ARN/genética , ARN Mensajero/metabolismoRESUMEN
Chemokines are signaling proteins that are secreted to induce chemotaxis during an immunological response. However, the functions of transmembrane-type chemokine-like factor (CKLF) and the CMTM (CKLF-like MARVEL transmembrane domain containing) protein family remain to be determined. In this study, we focused on the testis-specific mouse CMTM gene cluster (Cmtm1, Cmtm2a and Cmtm2b) and generated CRISPR/Cas9-mediated mutant mice to examine their physiological functions. Although Cmtm1 mutant mice were fertile, Cmtm2a and Cmtm2b double mutant mice had defects in male fertility due to impaired sperm function. We found that co-expression of sperm membrane proteins CMTM2A and CMTM2B is required for male fertility and affects the localization of the sperm membrane protein ADAM3 in regulating sperm fertilizing ability.
Asunto(s)
Proteínas ADAM/metabolismo , Quimiocinas/metabolismo , Fertilidad , Proteínas con Dominio MARVEL/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Represoras/metabolismo , Espermatozoides/metabolismo , Animales , Quimiocinas/genética , Proteínas con Dominio MARVEL/genética , Masculino , Ratones Noqueados , Ratones Mutantes , Familia de Multigenes , Especificidad de Órganos , Unión Proteica , Transporte de Proteínas , Proteínas Represoras/genética , Cabeza del Espermatozoide/metabolismo , Testículo/metabolismoRESUMEN
In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility.
Asunto(s)
Fertilidad/genética , Metalotioneína/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Azoospermia/congénito , Azoospermia/genética , Azoospermia/metabolismo , Células COS , Chlorocebus aethiops , Masculino , Meiosis/genética , Metalotioneína/genética , Ratones , Ratones Noqueados , Espermatocitos/metabolismo , Espermatogonias/metabolismoRESUMEN
Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.
Asunto(s)
Dineínas/metabolismo , Proteínas/genética , Motilidad Espermática , Espermatozoides/fisiología , Adenosina Trifosfato/metabolismo , Animales , Axonema/metabolismo , Chlamydomonas/metabolismo , Cilios/metabolismo , Cruzamientos Genéticos , Citoesqueleto/metabolismo , Femenino , Flagelos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Homocigoto , Humanos , Masculino , Ratones , Microtúbulos/metabolismo , Mutación , Proteínas/fisiología , Espermátides/metabolismo , Testículo/metabolismoRESUMEN
We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4+ T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Inmunoprecipitación de Cromatina/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética/métodos , ARN Guía de Kinetoplastida/genética , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Proteína 9 Asociada a CRISPR/genética , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.
Asunto(s)
Sistemas CRISPR-Cas , Fertilidad/genética , Edición Génica , Regulación de la Expresión Génica/fisiología , Testículo/metabolismo , Animales , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Noqueados , TranscriptomaRESUMEN
Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.
Asunto(s)
Fertilidad/genética , Testículo/metabolismo , Animales , Evolución Biológica , Sistemas CRISPR-Cas , Femenino , Fertilización , Ingeniería Genética , Genómica , Masculino , Ratones , Ratones Noqueados , EspermatogénesisRESUMEN
A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 (PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848GâA/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models.
Asunto(s)
Proteínas de Homeodominio/genética , Infertilidad Masculina/metabolismo , Mutación Puntual , Teratozoospermia/metabolismo , Proteínas de Transporte Vesicular/genética , Animales , Secuencia de Bases , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Exones , Edición Génica , Proteínas de Homeodominio/fisiología , Homocigoto , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Transgénicos , Eliminación de Secuencia , Teratozoospermia/genética , Proteínas de Transporte Vesicular/fisiologíaRESUMEN
Mammalian chromosomes form a hierarchical structure within the cell nucleus, from chromatin loops, megabase (Mb)-sized topologically associating domains (TADs) to larger-scale A/B compartments. The molecular basis of the structures of loops and TADs has been actively studied. However, the A and B compartments, which correspond to early-replicating euchromatin and late-replicating heterochromatin, respectively, are still relatively unexplored. In this review, we focus on the A/B compartments, discuss their close relationship to DNA replication timing (RT), and introduce recent findings on the features of subcompartments revealed by detailed classification of the A/B compartments. In doing so, we speculate on the structure, potential function, and developmental dynamics of A/B compartments and subcompartments in mammalian cells.
RESUMEN
Cell death and proliferation are at a glance dichotomic events, but occasionally coupled. Caspases, traditionally known to execute apoptosis, play non-apoptotic roles, but their exact mechanism remains elusive. Here, using Drosophila intestinal stem cells (ISCs), we discovered that activation of caspases induces massive cell proliferation rather than cell death. We elucidate that a positive feedback circuit exists between caspases and JNK, which can simultaneously drive cell proliferation and cell death. In ISCs, signalling from JNK to caspases is defective, which skews the balance towards proliferation. Mechanistically, two-tiered regulation of the DIAP1 inhibitor rpr, through its transcription and its protein localization, exists. This work provides a conceptual framework that explains how caspases perform apoptotic and non-apoptotic functions in vivo and how ISCs accomplish their resistance to cell death.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retroalimentación , Proteínas Inhibidoras de la Apoptosis/metabolismo , Muerte Celular , Drosophila/metabolismo , Caspasas/metabolismo , Proliferación Celular/genética , Células Madre/metabolismoRESUMEN
Epigenetic abnormalities are extremely widespread in cancer. Some of them are mere consequences of transformation, but some actively contribute to cancer initiation and progression; they provide powerful new biological markers, as well as new targets for therapies. In this review, we examine the recent literature and focus on one particular aspect of epigenome deregulation: large-scale chromatin changes, causing global changes of DNA methylation or histone modifications. After a brief overview of the one-dimension (1D) and three-dimension (3D) epigenome in healthy cells and of its homeostasis mechanisms, we use selected examples to describe how many different events (mutations, changes in metabolism, and infections) can cause profound changes to the epigenome and fuel cancer. We then present the consequences for therapies and briefly discuss the role of single-cell approaches for the future progress of the field.
RESUMEN
Calreticulin (CALR) exon 9 frameshift mutations, commonly detected in essential thrombocythemia (ET) and primary myelofibrosis patients, activate signal transducer and activator of transcription (STAT) proteins in the presence of Myeloproliferative Leukemia Virus (MPL) and induce ET in vivo. Loss of the KDEL motif, an endoplasmic reticulum retention signal, and generation of many positively charged amino acids (AAs) in the mutated C-terminus are thought to be important for disease induction. To test this hypothesis, we generated mice harboring a Calr frameshift mutation using the CRISPR/Cas9 system. Deletion of 19-base pairs in exon 9 (c.1099-1117del), designated the del19 mutation, induced loss of the KDEL motif and generated many positively charged AAs, similar to human mutants. Calr del19 mice exhibited mild thrombocytosis, slightly increased megakaryocytes, and mild splenomegaly. In vitro experiments revealed that the murine CALR del19 mutant had a weaker ability to combine with murine MPL than the human CALR del52 mutant. Consequently, STAT5 activation was also very weak downstream of the murine mutant and murine MPL, and may be the reason for the mild disease severity. In summary, loss of the KDEL motif and positively charged AAs in the C-terminus of CALR is insufficient for MPL binding and ET development.
Asunto(s)
Calreticulina/genética , Trombocitosis/etiología , Animales , Humanos , Ratones , MutaciónRESUMEN
Sperm entry in mammalian oocytes triggers intracellular Ca2+ oscillations that initiate resumption of the meiotic cell cycle and subsequent activations. Here, we show that phospholipase C zeta 1 (PLCζ1) is the long-sought sperm-borne oocyte activation factor (SOAF). Plcz1 gene knockout (KO) mouse spermatozoa fail to induce Ca2+ changes in intracytoplasmic sperm injection (ICSI). In contrast to ICSI, Plcz1 KO spermatozoa induced atypical patterns of Ca2+ changes in normal fertilizations, and most of the fertilized oocytes ceased development at the 1-2-cell stage because of oocyte activation failure or polyspermy. We further discovered that both zona pellucida block to polyspermy (ZPBP) and plasma membrane block to polyspermy (PMBP) were delayed in oocytes fertilized with Plcz1 KO spermatozoa. With the observation that polyspermy is rare in astacin-like metalloendopeptidase (Astl) KO female oocytes that lack ZPBP, we conclude that PMPB plays more critical role than ZPBP in vivo. Finally, we obtained healthy pups from male mice carrying human infertile PLCZ1 mutation by single sperm ICSI supplemented with Plcz1 mRNA injection. These results suggest that mammalian spermatozoa have a primitive oocyte activation mechanism and that PLCζ1 is a SOAF that ensures oocyte activation steps for monospermic fertilization in mammals.
Asunto(s)
Fertilización/genética , Fosfoinositido Fosfolipasa C/genética , Espermatozoides/metabolismo , Animales , Femenino , Masculino , Metaloproteasas/genética , Metaloproteasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Mutación , Oocitos/metabolismo , Oocitos/fisiología , Fosfoinositido Fosfolipasa C/metabolismo , Espermatozoides/fisiologíaRESUMEN
In mammalian cells, genome editing with the single guide RNA (sgRNA)/Cas9 complex allows for high targeting efficiency within a relatively short time frame with the added benefits of being low cost and easy to design. sgRNA/Cas9-mediated editing in mouse zygotes has accelerated the analysis of gene functions and the generation of mouse models of human diseases. Despite the benefits, this method still suffers from several problems, such as mosaicism in the founder generation which complicates genotyping and phenotypical analyses, and the low efficiency of more complicated genome editing. Thus, we recently established the system for genome editing in embryonic stem (ES) cells and its application for chimeric analysis in mice. In this section, we introduce the procedure for genome editing in mouse zygotes and ES cells.
Asunto(s)
Células Madre Embrionarias/citología , Edición Génica/métodos , ARN Guía de Kinetoplastida/genética , Cigoto/citología , Animales , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Quimera/genética , Ratones , Microinyecciones , Mosaicismo , Fenotipo , Plásmidos/genéticaRESUMEN
The paternal-allele-specific methylation of the Igf2/H19 imprinting control region (ICR) is established during gametogenesis and maintained throughout development. To elucidate the requirement of the germline passage in the maintenance of the imprinting methylation, we established a system introducing a methylated or unmethylated ICR-containing DNA fragment (ICR-F) into the paternal or maternal genome by microinjecting into the paternal or maternal pronucleus of fertilized eggs, and traced the methylation pattern in the ICR-F. When the ICR-F was injected in a methylated form, it was demethylated approximately to half degree at blastocyst stage but was almost completely remethylated at 3 weeks of age. In the case of the unmethylated form, the ICR-F remained unmethylated at the blastocyst stage, but was almost half-methylated at 3 weeks of age. Interestingly, the paternally injected ICR-F was highly methylated compared with maternally injected ICR-F at 3 weeks of age, partially mimicking the endogenous methylation pattern. Moreover, introduction of mutations in the CTCF (CCCTC binding factor) binding sites of the ICR-F, which are known to be important for the maintenance of hypomethylated maternal ICR, induced hypermethylation of the mutated ICR-F in both paternal and maternal pronuclear injected 3-week-old mice. Our results suggest the presence of a protection-against-methylation activity of the CTCF binding site in establishing the preferential paternal methylation during post-fertilization development and the importance of germline passage in the maintenance of the parental specific methylation at H19 ICR.
Asunto(s)
Impresión Genómica/genética , ARN Largo no Codificante/genética , Animales , Fragmentación del ADN , Metilación de ADN/genética , Femenino , Gametogénesis/genética , Genoma/genética , Masculino , Ratones Endogámicos ICR , Ratones Endogámicos , Microinyecciones , Terapia de Reemplazo Mitocondrial , Mutación , CigotoRESUMEN
Cytochrome P450, family 3, subfamily A (CYP3A) enzymes metabolize approximately 50% of commercially available drugs. Recently, we developed fully humanized transchromosomic (Tc) CYP3A mice with the CYP3A cluster including CYP3A4, CYP3A5, CYP3A7, and CYP3A43. Our humanized CYP3A mice have the CYP3A5*3 (g.6986G) allele, resulting in the almost absence of CYP3A5 protein expression in the liver and intestine. To produce model mice for predicting CYP3A5's contribution to pharmacokinetics, we performed a single-nucleotide polymorphism (SNP) modification of CYP3A5 (g.6986G to A, *3 to *1) on the CYP3A cluster using genome editing in both mouse ES cells and fertilized eggs, and produced humanized CYP3A5*1 mice recapitulating the CYP3A5*1 carrier phenotype in humans. The humanized CYP3A mouse with CYP3A5*1 is the first Tc mouse for predicting the SNP effect on pharmacokinetics in humans. The combination of Tc technology and genome editing enables the production of useful humanized models that reflect humans with different SNPs.
Asunto(s)
Citocromo P-450 CYP3A/genética , Edición Génica , Modelos Animales , Farmacogenética/métodos , Polimorfismo de Nucleótido Simple , Animales , Animales Modificados Genéticamente , Humanos , RatonesRESUMEN
Sperm-egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell-cell adhesion. However, the mechanism of IZUMO1 recognition by JUNO has remained elusive. Here we report the crystal structure of mouse JUNO, at 2.3 Å resolution. A structural comparison of JUNO with the FRs revealed that JUNO and the FRs have similar overall structures, but JUNO lacks the folate-binding pocket, thereby explaining the inability of JUNO to bind folate. Further complementation of Juno knockout eggs with mutant Juno messenger RNAs revealed that the conserved, surface-exposed tryptophan residue of JUNO is required for sperm binding and fertilization. Our structure-based in vivo functional analyses provide a framework towards a mechanistic understanding of mammalian gamete recognition.
Asunto(s)
Fertilización/fisiología , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular , Cristalografía por Rayos X , Proteínas del Huevo , Femenino , Receptor 1 de Folato/química , Receptor 1 de Folato/metabolismo , Células HEK293 , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/genética , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Noqueados , Mutación , Receptores de Superficie Celular/genética , Interacciones Espermatozoide-Óvulo/fisiología , Triptófano/genética , Triptófano/metabolismoRESUMEN
Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.