Project Overview

Conventional meat production accounts for 15-24% of greenhouse emissions and the demand for meat is only increasing. Due to the environmental burden of factory farming, there is an urgent need for meat-based protein alternatives. One promising method to meet this demand is through cellular agriculture and cultured meat, with the purpose of producing meat products from animal cells in vitro. However, this solution has been hindered since the beginning due to high experimental cost and scale-up difficulties. This project aims to address these issues by genetically modifying bovine satellite cells (BSCs) such that they can divide indefinitely without entering senescence, thereby creating an immortal cell line. As a result, less primary bovine cells would be needed, thereby reducing costs.

We intend to achieve this goal by using CRISPR, transposons, or Recombinase Mediated Cassette Exchange (RMCE) to insert genes of interest –reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4)– into the bovine genome. TERT produces the catalytic component of telomerase, an enzyme that counteracts telomere shortening by adding repeating fragments of DNA to the ends of the telomeres. CDK4 has been shown to decrease senescence by preventing the stalling of a cell before it reaches the S-phase of the cell cycle.

Our specific aims are as follows:

Specific Aim 1: Transfect the TERT and CDK4 genes into BSCs and confirm production of the corresponding proteins.

Specific Aim 2: Assess the ability of TERT and CDK4 to enhance the proliferation rate of BSCs and extend the number of generations they can proliferate for.

This method has proven effective in the immortalization of other cell types, but has yet to be applied to bovine satellite cells. We will quantify our success by sequencing the genetically modified cells to identify our genes of interest, the subsequent proteins, and assess how long the cells can divide under in vitro cell culture conditions. Successful completion of this project would be a significant step towards the production of scalable cultured meat, which would have a strong positive impact on the accessibility, ethical merit, and ecological sustainability of meat consumption.

Our project can be summarized with the following figures:

Experiments

E. coli culture

Four different strains of E. coli were cultured in LB broth for one day. These included pb Rosa 26-Imm, sgRNA2, sgRNA4, and sgRNA5, representing our immortalization plasmid and three CRISPR plasmids with slightly different guide RNAs. The E. coli was stored in a -80ºC freezer, thawed, and placed in a tube with 30 mL LB and 30 uL antibiotic. These tubes were placed in a shaking incubator set to 37ºC and 250 rpm, where they remained overnight.

Miniprep

After the E. coli were cultured, the plasmids were extracted from the E. Coli using a ThermoScientific GeneJET Plasmid Miniprep Kit. The cells were resuspended, lysed, and neutralized. The DNA was then bound to a column and eluted, resulting in tubes of the purified DNA.

Bovine Satellite Cell Culture

Bovine Satellite cells (BSCs) were cultured on laminin-coated tissue-culture plastic and grown with BSC GM, containing DMEM, 20% Fetal Bovine Serum, 1 ng/mL Fibroblast growth factor 2 (FGF-2), and 1% antibiotic/antimycotic. These cells were isolated previously and cryopreserved in liquid nitrogen. BSC’s were thawed and plated onto a 6-well plate at a density of 250,000 cells with 4.8 uL laminin per well.

Staining

To confirm the myogenicity of the bovine cells, two characterization stains were performed. These were Pax7 and MF20. Both were also stained using DAPI for comparison. Pax7 is a satellite cell marker, MF20 stains for Myosin Heavy Chain, and DAPI stains for cell nuclei.

Transfection

The cells were transfected using the reagent Lipofectamine™ 3000. The purified plasmids acquired from the elution (sgRNA 2, 4, and 5) were each added to a tube with opti-mem, P3000 reagent, and pb Rosa 26-Imm. A mixture of opti-mem and Lipofectamine™ 3000 was then added to this mixture, allowing for the formation of mycells. This was mixed and left to sit for 15 minutes at room temperature. This mixture was then added to the cell culture, without the addition of FBS-containing BSC GM. This was to avoid any conflicts and interactions between FBS and the transfection process.

Sleeping Beauty

Following CRISPR, the Sleeping Beauty transposon method was implemented for immortalization. Sleeping Beauty is better equipped at handling larger segments of DNA, at the expense of control. This was approximately a 24-day process. We have designed primers using Benchling. We then performed PCR using these primers and confirmed the success of this process through gel electrophoresis. We purified the resulting PCR fragments and used them to perform a Gibson Reaction to assemble our plasmid. We then transformed and sequenced E. coli using these constructs. After this was complete, we followed the same steps we did previously by culturing the E. coli in LB broth, performing a miniprep on the resulting E. coli, and using Lipofectamine to transfect the resulting purified DNA into Bovine Satellite Cells in culture.

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