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Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

ABSTRACT: Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.

SUBMITTER: Parreno J

PROVIDER: S-EPMC4749754 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

Parreno Justin J Delve Elizabeth E Andrejevic Katarina K Paez-Parent Sabrina S Wu Po-Han PH Kandel Rita R

Cartilage 20160101 1

Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using v ...[more]

PMID: 26958320

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Project description:BackgroundSmall RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches.ResultsWe have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents.ConclusionsOur streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

Dataset Information

Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

Publications

Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

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