Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.
CRISPR
Cas9
Drosophila
Drosophila oogenesis
GFP
Gene tagging
LEM-domain proteins
Nuclear lamina
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
30
1
2023
pubmed:
31
1
2023
medline:
2
2
2023
Statut:
ppublish
Résumé
The Drosophila ovary represents an outstanding model for investigating tissue homeostasis. Females continuously produce oocytes throughout their lifetime. However, as females age, fecundity declines, in part, due to changes in ovarian niche function and germline stem cell (GSC) homeostasis. Understanding the dynamics of GSC maintenance will provide needed insights into how coordinated tissue homeostasis is lost during aging. Critical regulators of GSC maintenance are proteins that reside in the nuclear lamina (NL), including the NL proteins emerin and Barrier-to-Autointegration Factor (BAF). Continued investigation of how emerin, BAF, and other NL proteins contribute to GSC function depends upon the availability of antibodies for NL proteins, a limiting resource. In this chapter, we discuss strategies for using clustered regularly interspaced short palindromic repeats (CRISPR) genomic editing to produce endogenously tagged NL genes to circumvent this obstacle, using the generation of the gfp-baf allele as an example. We describe strategies for validation of tagged alleles. Finally, we outline methods for immunohistochemical analysis of resulting tagged-NL proteins.
Identifiants
pubmed: 36715902
doi: 10.1007/978-1-0716-2970-3_6
doi:
Substances chimiques
Drosophila Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
109-134Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM087341
Pays : United States
Organisme : NIA NIH HHS
ID : R21 AG072397
Pays : United States
Organisme : NCRR NIH HHS
ID : S10 RR025439
Pays : United States
Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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