Reverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest. Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation. Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued. The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.

中文翻译：

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连接和限制酶独立克隆技术：一种替代传统方法的方法，用于在流感反向遗传学系统中克隆难以克隆的基因片段。

反向遗传学在世界各地的许多实验室中得到应用，能够创建具有所需基因型或表型的定制流感病毒。然而，该过程并非完美无缺，在将流感基因片段克隆到反向遗传学载体（pHW2000、pHH21、pCAGGS）的过程中仍然存在困难。反向遗传学首先制作流感基因片段的 cDNA 拷贝并将它们克隆到双向 (pHW2000) 或单向质粒 (pHH21、pCAGGS)，然后将重组质粒转染到 HEK-293 T 或任何其他合适的允许转染的细胞。然而，如果采用常规方法，许多禽流感病毒野外分离株的基因片段中存在内部限制性位点，这使得克隆过程变得困难。更远，细菌中含有流感基因的质粒（尤其是聚合酶 Basic 2 和聚合酶 Basic 1 基因；PB2 和 PB1）的遗传不稳定性也会导致细菌基因组序列错误地掺入目标流感基因。在此，我们报告了一种简单有效的连接和限制酶非依赖性 (LREI) 克隆方法，用于将流感基因片段克隆到 pHW2000 载体中。该方法包括扩增引物，然后使用诱饵质粒、DpnI 消化和转化对引物进行 PCR 扩增。难克隆基因：PB2 of A/chicken/Bangladesh/23527/2014 (H9N2)和PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02。使用我们的 LREI 方法将 05YGYXG023-P/2015 (H5N6) 和 A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) 克隆到 pHW2000 中，随后拯救了重组病毒。LREI 克隆程序代表了一种用于克隆流感基因片段的替代策略，这些片段具有反向遗传学中使用的酶的内部限制性位点。此外，可以通过在较低温度下培养重组细菌培养物来缓解细菌遗传不稳定的问题。该技术可用于使用通用引物克隆任何流感基因片段，这将有助于快速产生流感病毒并促进流感研究和疫苗开发。LREI 克隆程序代表了一种用于克隆流感基因片段的替代策略，这些片段具有反向遗传学中使用的酶的内部限制性位点。此外，可以通过在较低温度下培养重组细菌培养物来缓解细菌遗传不稳定的问题。该技术可用于使用通用引物克隆任何流感基因片段，这将有助于快速产生流感病毒并促进流感研究和疫苗开发。LREI 克隆程序代表了一种用于克隆流感基因片段的替代策略，这些片段具有反向遗传学中使用的酶的内部限制性位点。此外，可以通过在较低温度下培养重组细菌培养物来缓解细菌遗传不稳定的问题。该技术可用于使用通用引物克隆任何流感基因片段，这将有助于快速产生流感病毒并促进流感研究和疫苗开发。