Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.
Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.
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DOI：
10.1007/978-1-0716-2970-3_6
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发表时间：
2023-01-01
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影响因子：
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通讯作者：
Geyer, Pamela K
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文献类型：
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作者：
Duan, Tingting;Rodriguez-Tirado, Felipe;Geyer, Pamela K
果蝇卵巢是研究组织稳态的一个杰出模型。雌性在一生中不断产生卵母细胞。然而，随着女性年龄的增长，生殖力下降，部分原因是卵巢生态位功能和种系干细胞（GSC）稳态的变化。了解GSC维持的动力学将为了解组织在衰老过程中如何失去协调的稳态提供必要的见解。GSC维持的关键调节因子是位于核层（NL）中的蛋白质，包括NL蛋白emerin和自整合障碍因子（BAF）。emerin、BAF和其他NL蛋白如何促进GSC功能的持续研究取决于NL蛋白抗体的可用性，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，讨论了使用集群规则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基因以规避这一障碍的策略。我们描述了验证标记等位基因的策略。最后，我们概述了免疫组织化学方法分析所得标记的nl蛋白。
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.