An Animal Model for Genetic Analysis of Multi-Gene Families: Cloning and Transgenesis of Large Tandemly Repeated Histone Gene Clusters.
Methods Mol Biol
; 1832: 309-325, 2018.
Article
en En
| MEDLINE
| ID: mdl-30073535
ABSTRACT
Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Histonas
/
Familia de Multigenes
/
Clonación Molecular
/
Técnicas de Transferencia de Gen
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Secuencias Repetidas en Tándem
/
Drosophila melanogaster
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos