Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target

IF 3.5 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology and Bioengineering Pub Date : 2024-07-08 DOI:10.1002/bit.28801

Kyuhee Cho, Jae Seong Lee

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引用次数: 0

Abstract

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

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重新审视基因组热点的影响：作为热点和工程目标的 C12orf35 基因座

传统的中国仓鼠卵巢（CHO）细胞系开发是基于转基因的随机整合（RI），这会导致克隆变异和随后的大规模克隆筛选。因此，将转基因特异位点整合（SSI）到基因组热点上最近成为细胞系开发的另一种方法。然而，热点位点形成的具体机制仍不清楚。在本研究中，我们旨在通过对编码荧光报告蛋白的转基因进行位点特异性整合（RI），产生着陆垫（LP）细胞系，其两侧是重组位点，以促进重组酶介导的盒式交换。与在 CHO 基因组热点 C12orf35 基因座上的相同报告基因 LP 构建的定向整合株相比，自发 C12orf35 基因座缺失的、表达高水平报告基因的基于 RI 的 LP 细胞系表现出相似的报告基因荧光蛋白水平。此外，在具有保守的 C12orf35 的基于 RI 的 LP 细胞系中，C12orf35 基因座基因 Resf1 的敲除（KO）增加了报告表达水平，与 C12orf35 基因座缺失的细胞系相当。这些结果表明，基因组热点 C12orf35 基因座的 SSI 对高水平转基因表达的影响是由 C12orf35 干扰引起的。与 C12orf35 KO 不同的是，在特定基因位点的其他著名热点位点，包括 Fer1L4、Hprt1、Adgrl4、Clcc1、Dop1b 和 Ddc，进行 KO 并没有增加转基因的表达。总之，我们的研究结果表明，C12orf35 是一个很有前途的工程目标，也是基于 SSI 的细胞系开发的热点。

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来源期刊

Biotechnology and Bioengineering 工程技术-生物工程与应用微生物

CiteScore

7.90

自引率

5.30%

发文量

280

审稿时长

2.1 months

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