Wittmann Jürgen
Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger-Center of Molecular Medicine (NFZ), Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
Sci Rep. 2025 Mar 11;15(1):8348. doi: 10.1038/s41598-025-93222-y.
The pBMN-I-GFP plasmid is a widely used retroviral vector for producing retroviral particles, utilized by thousands of laboratories worldwide. However, we observed that E. coli transformed with pBMN-I-GFP failed to grow on selective LB agar plates containing ampicillin or carbenicillin, in contrast to similar retroviral vectors. Multiple attempts to optimize growth conditions were unsuccessful. Sequencing, contrary to the available reference sequence, revealed an inversion of the beta-lactamase (bla) gene and part of its promoter, likely disrupting bla expression and, consequently, antibiotic resistance. To address this, we corrected the orientation of the ampicillin resistance gene and its promoter in a new plasmid, prBMN-I-EGFP. This modification restored robust growth of E. coli transformed with this plasmid on selective plates, confirming the essential role of an intact bla promoter for antibiotic resistance. Additionally, retroviral functionality tests in murine cell lines showed that prBMN-I-EGFP exhibited transfection and infection efficiencies comparable to the original pBMN-I-GFP. These findings underscore the importance of thorough sequence verification for commonly used plasmids and present an improved version of pBMN-I-GFP.
pBMN-I-GFP质粒是一种广泛用于生产逆转录病毒颗粒的逆转录病毒载体，全球数千个实验室都在使用。然而，我们观察到，与类似的逆转录病毒载体相比，用pBMN-I-GFP转化的大肠杆菌在含有氨苄青霉素或羧苄青霉素的选择性LB琼脂平板上无法生长。多次尝试优化生长条件均未成功。测序结果与可用的参考序列相反，显示β-内酰胺酶（bla）基因及其部分启动子发生了倒置，这可能破坏了bla的表达，从而导致抗生素耐药性丧失。为了解决这个问题，我们在一个新的质粒prBMN-I-EGFP中纠正了氨苄青霉素抗性基因及其启动子的方向。这种修饰恢复了用该质粒转化的大肠杆菌在选择性平板上的强劲生长，证实了完整的bla启动子对抗生素耐药性的重要作用。此外，在小鼠细胞系中的逆转录病毒功能测试表明，prBMN-I-EGFP的转染和感染效率与原始的pBMN-I-GFP相当。这些发现强调了对常用质粒进行全面序列验证的重要性，并展示了pBMN-I-GFP的改进版本。
