Yanagi Katherine S, Lehrbach Nicolas
Basic Sciences Division, Fred Hutch Cancer Center, Seattle, Washington, United States.
MicroPubl Biol. 2024 May 29;2024. doi: 10.17912/micropub.biology.001230. eCollection 2024.
Transgenic animals are an invaluable tool in model organism genetics. The ease of modifying the genome through high-copy integration of transgenes facilitates the investigation of diverse and fundamental biological processes. However, generation of new multicopy integrated transgenes is limited by the time and labor cost. Further, many transgenes are integrated using non-specific DNA damaging agents. These DNA damaging agents cause unwanted mutations during the integration process and may have deleterious effects. A recently described method for CRISPR/Cas9-based integration of multicopy transgenes at safe harbor loci using Fluorescent Landmark Interference (FLInt) greatly increases the efficiency of multicopy transgene integration and mitigates issues related to off-target mutagenesis during integration. rescue is a simple and widely used phenotypic marker in transgenesis and genome engineering. To streamline generation of multicopy transgenes via FLInt, we have generated a set of strains suitable for FLInt-mediated integration of transgenes using rescue of the mutant phenotype to select transgenic animals. We demonstrate the utility of this approach and outline a protocol that uses rescue as a selection marker for streamlined integration of multicopy transgenes at safe harbor loci.
转基因动物是模式生物遗传学中一种非常有价值的工具。通过转基因的高拷贝整合来修饰基因组的便利性，有助于对各种基本生物学过程进行研究。然而，新的多拷贝整合转基因的产生受到时间和劳动力成本的限制。此外，许多转基因是使用非特异性DNA损伤剂进行整合的。这些DNA损伤剂在整合过程中会导致不必要的突变，并且可能产生有害影响。最近描述的一种基于CRISPR/Cas9的方法，利用荧光标记干扰（FLInt）在安全位点整合多拷贝转基因，极大地提高了多拷贝转基因整合的效率，并减轻了整合过程中与脱靶诱变相关的问题。拯救是转基因和基因组工程中一种简单且广泛使用的表型标记。为了简化通过FLInt产生多拷贝转基因的过程，我们已经生成了一组菌株，这些菌株适合于利用突变体表型的拯救来选择转基因动物，从而通过FLInt介导转基因的整合。我们展示了这种方法的实用性，并概述了一种方案，该方案使用拯救作为选择标记，用于在安全位点简化多拷贝转基因的整合。
