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. 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. 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 process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). 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. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
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