Book chapter
Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes
Drosophila Oogenesis, Vol.2626, pp.109-134
Methods in molecular biology (Clifton, N.J.), 2626, Humana
2023
DOI: 10.1007/978-1-0716-2970-3_6
PMID: 36715902
Abstract
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.
Details
- Title: Subtitle
- Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes
- Creators
- Tingting Duan - University of IowaFelipe Rodriguez-Tirado - Department of Biochemistry and Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USAPamela K Geyer - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, USA. pamela-geyer@uiowa.edu
- Resource Type
- Book chapter
- Publication Details
- Drosophila Oogenesis, Vol.2626, pp.109-134
- Publisher
- Humana; New York, NY
- Series
- Methods in molecular biology (Clifton, N.J.); 2626
- DOI
- 10.1007/978-1-0716-2970-3_6
- PMID
- 36715902
- ISSN
- 1940-6029
- eISSN
- 1940-6029
- Language
- English
- Date published
- 2023
- Academic Unit
- Molecular Physiology and Biophysics; Obstetrics and Gynecology; Biochemistry and Molecular Biology
- Record Identifier
- 9984363741002771
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