A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system.

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 µg/mL and 7 µg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Genome Editing: A Comparative Study on the Efficiency of CRISPR/Cas9 Nuclease Versus Nickase Using HIV as a Model System

@#Introduction: CRISPR/Cas9 nuclease has gained popularity as a genome editing tool due to its straight-forward mechanism. However, there are concerns that CRISPR nuclease would cause off-target and toxicity. The CRISPR/ Cas9 D10A nickase was designed to enhance genome editing. Nevertheless, this raised the question of whether the efficiency of nickase is compromised compared to CRISPR/Cas9 nuclease. Targeting HIV genes, we investigated if CRISPR nuclease performed better than the nickase in efficacy and safety. Methods: CRISPR nucleases and nickases were designed to target Gag, Pol, Rev, Vif, Tat and LTR. HIV latently infected cell line, ACH-2, was transfected with the nucleases and nickases. Changes to viral load after CRISPR treatment was measured using p24 ELISA. Safety of nuclease and nickase was monitored using GFP expression with fluorescence microscopy and flow cytometry. Targeting two sites within the same gene, and targeting multiple genes concurrently were also studied to determine efficacy of CRISPR in reducing viral load. Results: A 44.9 to 68.1% and a 34.4 to 49.7% decrease in viral load was seen in CRISPR nuclease and nickase respectively. Microscopy and flow cytometry results showed that the nickase system was slightly toxic with a 0.31 to 0.7-fold cell death. There was a 34% decrease in viral load when two sites were targeted within a gene, and the largest decrease was seen when all the nucleases were combined, giving a 75.4% decrease in viral load at day 5. Conclusion: The knowledge gained from this study will be employed to improve genome editing in other disease models.

Screening of endophytic bacteria isolated from leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] for cytokinin-like compounds.

UNLABELLED: Endophytic bacteria are harmless in most plant species; and known to boost the growth and development of the host plants probably by secreting growth hormones. The isolation, identification and screening of endophytic bacteria for the plant growth regulators like cytokinin are needed to get the leads for their applications in agriculture sector. We describe the isolation and identification of the bacterial endophytes from the leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] and their screening for cytokinin-like compounds. We isolated three endophytic bacteria from the leaves of G. procumbens collected from the forest research institute of Malaysia (FRIM). They were further identified using amplified 16S rRNA gene sequence based method of bacterial identification. The ethyl acetate extracts of the isolates-broth were analyzed using cucumber cotyledon greening bioassay (CCGB) to determine the presence of cytokinin-like compounds. Consequently, the bacterial putative endophytes were identified as Psuedomonas resinovorans, Paenibacillus polymaxa, and Acenitobacter calcoaceticus. Broth-extracts from two (Psuedomonas resinovorans and Paenibacillus polymaxa) of the three putative bacterial endophytes show the positive results in their screening for cytokinin-like compounds using CCGB. Thus, we hypothesize that the bacterial putative endophytes of G. procumbens that produce cytokinin-like compounds might have a role in the growth and development of G. procumbens. ABBREVIATIONS: CCGB - Cucumber cotyledon greening bioassay, rDNA - Ribosomal DNA, K12, BAP - 6-Benzylaminopurine, Db1, MSA - Multiple sequence alignment. 8081.