RESUMO
Understanding the contributions of mitochondrial genetics to disease pathogenesis is facilitated by a new and unique model-the mitochondrial-nuclear exchange mouse. Here we report the rationale for their development, the methods used to create them, and a brief summary of how MNX mice have been used to understand the contributions of mitochondrial DNA in multiple diseases, focusing on cancer metastasis. Polymorphisms in mtDNA which distinguish mouse strains exert intrinsic and extrinsic effects on metastasis efficiency by altering epigenetic marks in the nuclear genome, changing production of reactive oxygen species, altering the microbiota, and influencing immune responses to cancer cells. Although the focus of this report is cancer metastasis, MNX mice have proven to be valuable in studying mitochondrial contributions to other diseases as well.
Assuntos
Mitocôndrias , Neoplasias , Camundongos , Animais , Mitocôndrias/genética , Mitocôndrias/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Polimorfismo Genético , Espécies Reativas de Oxigênio/metabolismo , Núcleo Celular/metabolismo , Neoplasias/patologiaRESUMO
Generating a transgenic or gene-modified mouse requires the introduction of exogenous reagents into an early-stage embryo. The mouse one-cell embryo or zygote possesses two pronuclei, representing the genetic contribution of the sperm and oocyte. Traditional transgenic mice are generated by injecting a DNA solution containing a purified transgene construct into the male pronucleus, generally the larger of the two pronuclei. Similarly, gene-editing reagents such as ZFNs, TALENs, and CRISPR RNAs are introduced into zygotes in the same manner, making this technique applicable to a wide variety of projects. This chapter presents the procedures for pronuclear microinjection.
Assuntos
Núcleo Celular/genética , DNA/genética , Desenvolvimento Embrionário/genética , Camundongos Transgênicos/genética , Animais , Feminino , Edição de Genes , Masculino , Camundongos , Microinjeções , Oócitos/crescimento & desenvolvimento , Espermatozoides/crescimento & desenvolvimento , Transgenes/genética , Zigoto/crescimento & desenvolvimentoRESUMO
The ability to delete the function of an endogenous gene in the mouse was made possible by the development of embryonic stem (ES) cells, pluripotent cells that retain the ability to develop into all tissues of a developing embryo. The ability to genetically modify these cells followed, allowing targeted mutation of ES cells in vitro and the deletion of specific gene function. However, regardless of the simplicity or complexity of the genetic modification, all ES cells require injection into host embryos to establish pregnancies and result in chimeric mice. Blastocysts are commonly used as the host embryos for this purpose, as it is relatively easy to inject cells into the blastocoel cavity of the developing embryo. This chapter describes the procedure for injection of ES cells into blastocyst stage embryos for the generation of knockout mice.
Assuntos
Transferência Embrionária/métodos , Células-Tronco Embrionárias , Microinjeções/métodos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Quimera/genética , Feminino , Camundongos , Camundongos Knockout , GravidezRESUMO
Embryo transfer surgery is an essential step in the process of generating gene-modified mice, regardless of whether the embryos were modified by DNA, RNA CRISPR components, or embryonic stem cells, or whether they were microinjected or electroporated. Transfer is also a necessary step for assisted reproductive techniques such as rederivation and reanimation by in vitro fertilization. The manipulated embryos must be returned to the reproductive tract of a pseudopregnant recipient female mouse, wherein the transplanted embryos develop to term. Embryos may be transferred to either the oviduct or uterine horn, depending upon the developmental status of the embryos and the stage of the recipient. This chapter will describe the process of transferring embryos surgically to a recipient female.
Assuntos
Transferência Embrionária/métodos , Fertilização in vitro/métodos , Microinjeções/métodos , Animais , Sistemas CRISPR-Cas/genética , Implantação do Embrião/genética , Tubas Uterinas/cirurgia , Feminino , Humanos , CamundongosRESUMO
The CRISPR/Cas9 system has enjoyed enormous success and has now become the standard method of generating gene-modified mouse models. The tools for predicting the activity of CRISPR reagents in the mouse embryo are currently limited and not particularly accurate in predicting if a given reagent will be active. Given the time and cost of generating genetically modified mice, it is highly desirable to use CRISPR reagents that are known to be active in the mouse embryo. In this chapter, we provide a detailed procedure for empirically testing the activity of CRISPR reagents via electroporation into cultured preimplantation mouse embryos. This platform has proven to be rapid, efficient, and applicable to a variety of mouse strains, and can be used for assessing on- and off-target activity through a variety of molecular assays.
Assuntos
Blastocisto , Sistemas CRISPR-Cas/genética , Desenvolvimento Embrionário/genética , Edição de Genes/métodos , Animais , Camundongos , Zigoto/crescimento & desenvolvimentoRESUMO
OBJECTIVE: To evaluate the reliability of the Focal-Point slide profiler (TriPath Care Technologies, Burlington, North Carolina, U.S.A.) in determing the absence of endocervical cells in conventional Pap smear slides. STUDY DESIGN: A consecutive series of conventional Pap smears, designated by FocalPoint as requiring no further review (NFR) and as lacking endocervical cells, was manually screened to determine the true presence or absence of endocervical cells. These results were compared to those obtained by FocalPoint. RESULTS: From January 1, 2000, to December 31, 2001, FocalPoint indicated that 797 NFR slides did not contain endocervical cells. In contrast, manual screening revealed that 504/797 (63.2%) did contain endocervical cells. CONCLUSION: The reliability of a negative FocalPoint determination for endocervical cells is limited. Manual screening of NFR slides designated by the instrument as lacking endocervical cells appears to be necessary.
Assuntos
Carcinoma/patologia , Colo do Útero/patologia , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Teste de Papanicolaou , Neoplasias do Colo do Útero/patologia , Esfregaço Vaginal/instrumentação , Esfregaço Vaginal/métodos , Feminino , Humanos , Reprodutibilidade dos TestesRESUMO
Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17ß-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.