Duan Tingting, Rodriguez-Tirado Felipe, Geyer Pamela K
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Department of Biochemistry and Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Methods Mol Biol. 2023;2626:109-134. doi: 10.1007/978-1-0716-2970-3_6.
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.
果蝇卵巢是研究组织稳态的一个杰出模型。雌性果蝇在其一生中持续产生卵母细胞。然而，随着雌性果蝇年龄增长，生育力会下降，部分原因是卵巢微环境功能和生殖系干细胞（GSC）稳态的变化。了解GSC维持的动态过程将为深入了解衰老过程中协调的组织稳态是如何丧失提供必要的见解。GSC维持的关键调节因子是位于核纤层（NL）中的蛋白质，包括NL蛋白emerin和屏障自整合因子（BAF）。对emerin、BAF和其他NL蛋白如何促进GSC功能的持续研究依赖于NL蛋白抗体的可用性，而这是一种有限的资源。在本章中，我们以gfp - baf等位基因的产生为例，讨论使用成簇规律间隔短回文重复序列（CRISPR）基因组编辑来产生内源性标记的NL基因以克服这一障碍的策略。我们描述了验证标记等位基因的策略。最后，我们概述了对所得标记NL蛋白进行免疫组织化学分析的方法。
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