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. 2022 Feb 25;9(3):91.
doi: 10.3390/bioengineering9030091.

Engineering Novel Lentiviral Vectors for Labelling Tumour Cells and Oncogenic Proteins

Seçkin Akgül  1   2 , Carolin Offenhäuser  1 , Anja Kordowski  1   3 , Bryan W Day  1   3   4
Affiliations

Engineering Novel Lentiviral Vectors for Labelling Tumour Cells and Oncogenic Proteins

Seçkin Akgül et al. Bioengineering (Basel). .

Abstract

Lentiviral vectors are unique and highly efficient genetic tools to incorporate genetic materials into the genome of a variety of cells whilst conserving biosafety. Their rapid acceptance made it necessary to improve existing protocols, including molecular engineering and cloning, production of purified lentiviral particles, and efficient infection of target cells. In addition to traditional protocols, which can be time-consuming, several biotechnology companies are providing scientists with commercially available lentiviral constructs and particles. However, these constructs are limited by their original form, tend to be costly, and lack the flexibility to re-engineer based on the ever-changing needs of scientific projects. Therefore, the current study organizes the existing methods and integrates them with novel ideas to establish a protocol that is simple and efficient to implement. In this study we, (i) generated an innovative site-directed nucleotide attachment/replacement and DNA insertion method using unique PCR primers, (ii) improved traditional methods by integrating plasmid clarification steps, (iii) utilized endogenous mRNA as a resource to construct new lentiviruses, and (iv) identified an existing purification method and incorporated it into an organized workflow to produce high-yield lentiviral particle collection. Finally, (v) we verified and demonstrated the functional validity of our methods using an infection strategy.

Keywords: genetic engineering; lentiviral particle; lentivirus; molecular cloning; plasmid; site-directed mutagenesis; tumour heterogeneity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The outline of the molecular cloning strategy to engineer novel lentiviral plasmids (A,B). Specific primers were designed to incorporate restriction sites in flanking regions of both vector (pLVX) and insert (E2-Crimson) (C). High-fidelity polymerase chain reaction enabled the production of linear DNA fragments (D). Restriction digestion with XhoI and EcoRI resulted in complementary sticky ends in the vector and insert (E). Ligation reaction allowed the specific fusion of two DNA fragments yielding a new lentiviral plasmid that carry the E2-Crimson fluorescent protein gene.
Figure 2
Figure 2
Generation of lentiviral plasmids with different fluorescent protein genes (A). Optimum template DNA and annealing temperature is determined to synthesize empty vector (pLVX). The most advantageous tested condition is indicated with a yellow asterisk (B). Minimal DNA template was used to synthesize insert (E2-Crimson). The most advantageous tested condition is indicated with a yellow asterisk (C). Restriction digestion with XhoI and EcoRI identified the newly engineered plasmids with correct DNA sizes (indicated by yellow arrows).
Figure 3
Figure 3
Outline of the molecular cloning strategy to introduce small peptides in wild type genes (A,B). Specific primers were designed to incorporate restriction sites in flanking regions of both vector (pLVX) and insert (EPHA3). HA-tag is added after the 20-aa signal peptide of EPHA3 gene (C). High-fidelity polymerase chain reaction enabled production of linear DNA fragments (D). Restriction digestion with XhoI and EcoRI resulted in complementary sticky ends in the vector and insert (E). Ligation reaction allowed specific fusion of two DNA fragments, yielding a new lentiviral plasmid that carries the HA-tagged EPHA3 gene.
Figure 4
Figure 4
Generation of new lentiviral plasmids allows HA-tagged proteins (A). Optimum annealing temperature is determined to synthesize insert (HA.EPHA3). The most advantageous tested condition is indicated with a yellow asterisk (B). Restriction digestion with XhoI and EcoRI identified the newly engineered pLVX.HA.EPHA3 plasmids with correct DNA sizes (indicated by an arrow). Abbreviations: U: undigested DNA; D: digested DNA.
Figure 5
Figure 5
Newly designed lentiviral particles are used in cell tracing and protein labelling (A). Lentiviral particles allowed expression of E2. Crimson and ZsYellow fluorescent proteins. Curved arrows indicate that pLVX-E2. Crimson-C1 and pLVX-ZsYellow-C1 lentiviral plasmids were prepared by using pLVX-AmCyan1-C1 as a vector backbone (B). Chimeric expression of five different fluorescent proteins in tumour cells that are infected with lentiviral particles (C). Lentiviral particles allowed expression of HA-tagged EPHA3 protein in PC-3 cells. The left lane is wild type (wt) PC-3 cells with undetectable EPHA3 and HA-tag expression. The right lane is PC-3 cells infected with HA.EPHA3 lentiviral particles. β-actin was used as a loading control (D). Cell membrane localisation of HA-tagged EPHA3 protein was tested by FACS analysis of nonpermeabilized cells. EPHA3 antibody successfully detected the recombinant EPHA3 protein.
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