Brian T Nguyen, Nora M Chapman, John C Mullican, Kristen M Drescher
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As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2000,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.\",\"authors\":\"Brian T Nguyen, Nora M Chapman, John C Mullican, Kristen M Drescher\",\"doi\":\"10.3791/66726\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). 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引用次数: 0
摘要
奈格勒氏滋养体的所有核糖体基因都保存在一个封闭的环状染色体外核糖体 DNA(rDNA)含元(CERE)中。虽然人们对 CERE 知之甚少,但对三种奈格勒氏滋养体的完整基因组序列分析清楚地表明,核基因组中没有 rDNA 单列子。此外,在 N. gruberi CERE 中绘制了一个单一的 DNA 复制源,支持了 CERE 独立于核基因组进行复制的假设。CERE 的这一特性表明,有可能利用改造的 CERE 将外来蛋白质引入奈格勒氏滋养体。作为探索在奈格勒氏滋养体中使用 CERE 作为载体的第一步,我们制定了一套方案,用克隆到 pGEM7zf+ (pGRUB)中的奈格勒氏滋养体 CERE 分子克隆转染奈格勒氏滋养体。转染后,pGRUB 可在 N. gruberi 滋养体中检测到至少 7 个传代以及包囊和外包囊。作为对照,滋养体转染了不含 N. gruberi 序列的骨干载体 pGEM7zf+(pGEM)。pGEM 在转染到 N. gruberi 滋养体后的第一次传代后就检测不到了,这表明它无法在真核生物体内复制。这些研究描述了奈格拉滋养体的转染方案,并证明 pGRUB 中的细菌质粒序列不会抑制转染 CERE 克隆的成功转染和复制。此外,该转染方案对于了解 CERE 在滋养体中复制的最小驱动序列以及确定非核糖体序列(NRS)中的调控区域至关重要。
Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.
All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.