RESUMO
With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.
Assuntos
Biotecnologia/métodos , Edição de Genes/métodos , Engenharia Genética/métodos , Nucleases de Dedos de Zinco/metabolismo , Biotecnologia/instrumentação , Mutação com Ganho de Função/genética , Deleção de Genes , Edição de Genes/instrumentação , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Introdução de Genes/instrumentação , Técnicas de Introdução de Genes/métodos , Engenharia Genética/instrumentação , Genoma de Planta/genética , Nucleases de Dedos de Zinco/genéticaRESUMO
In 1991, Soriano and coworkers isolated the ROSA26 locus in a gene-trap mutagenesis screening performed in mouse embryonic stem (ES) cells. The ubiquitous expression of ROSA26 in embryonic and adult tissues, together with the high frequency of gene-targeting events observed at this locus in murine ES cells has led to the establishment in the past 10 years of over 130 knock-in lines expressing successfully from the ROSA26 locus a variety of transgenes including reporters, site-specific recombinases and, recently, noncoding RNAs. Different strategies can be employed to drive transgene expression from the ROSA26 locus. This chapter provides an overview of the current methodologies used to generate ROSA26 knock-in lines and describes different approaches that exploit the ROSA26 gene to control expression of transgenes, including miRNAs, in a temporal, cell-type, and stage-specific fashion.
