October 29th Biweekly Report

PROJECT TITLE: Immortalization of a Bovine Satellite Cell Line

TEAM MEMBERS: Miles Arnett, Tina Guo, Addison Mirliani

PI / MENTOR: Andrew Stout

PROJECT DESCRIPTION: There is currently a significant need for alternative sources of meat-based protein, largely because of the environmental burden of factory farming. One promising method of meeting this need is cultured meat, which aims to produce meat products from cultured animal cells. However, the field has been hampered since its inception by problems with experimental cost and scale-up efficiency. 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. This would allow for much less initial investment in purchasing bovine cells for experimental purposes while also making it far easier to generate large numbers of them. We intend to achieve this goal by using CRISPR, transposons, or Recombinase Mediated Cassette Exchange (RMCE) to insert genes of interest into the BSCs, which promote the expression of telomerase reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4). This method has proven effective in the immortalization of other cell types, but has yet to be applied to BSCs. We will quantify our success by sequencing the cells we produce to identify our genes of interest and by assessing how long our cells can divide under in vitro cell culture relative to a control. Successful completion of this aim would be a significant step forward for efforts to produce cultured meat, which would have a strong positive impact on the accessibility, ethical merit, and ecological sustainability of meat consumption.

Progress/Gantt Chart here:

https://docs.google.com/spreadsheets/d/1D7nFiVtEUOjNkVYPmYEqijVvi_tf1B2CW6-9qNmdUwg/edit?usp=sharing

ENGINEERING DESIGN ELEMENTS:

What are the objectives of the project and the criteria for selecting them?

Our first objective in this project is to construct plasmids containing our genes of interest, TERT and CDK4, and transfect them into BSCs using CRISPR-Cas9 machinery. This is a continuation of the previous endeavor in which our PI attempted to immortalize these cells, and will provide a good baseline level of effectiveness for our ability to modify them. Our next objective will be to image and analyze our altered cells to assess whether or not our transfection was successful, which will inform our next steps.

If the transfection is unsuccessful, our next objective will be to assess another gene editing system, likely the Sleeping Beauty transposon method, which is generally better at editing in larger plasmids than CRISPR, at the cost of some control. We will also likely attempt to use Recombinase Mediated Cassette Exchange, another gene editing system, to give ourselves as many chances as possible. When a successful transfection has been achieved, our next objective will be to monitor the proliferation and survival of the edited cells in comparison with a control over a long period of time, as this is ultimately how we will determine if they have been immortalized. We will also likely sequence the edited cells and conduct protein quantification to detect our genes and proteins of interest.

What system, component, or process is to be designed?

This project aims to design a genetically-modified cell line that will improve the process of Bovine Satellite Cell culture. These modified cells will not senesce, and will therefore improve this process by allowing for more passaging and greater proliferation in cell culture. We will use software platforms that will allow us to make edits to genetic sequences and ultimately formulate a sequence of genes that will be incorporated into bovine DNA. Achieving this aim will also involve designing a process for transfecting relatively large immortalization genes into BSCs, which has yet to be done.

What need does it fulfill (clinical, research, etc.)?

While immortalized BSC lines would be most beneficial to researchers in the cellular agriculture industry, it’s cost effectiveness and applicability will appeal to a wide array of research fields outside of cultured meat, such as drug toxicity tests and gene therapy. Immortalized cell lines have a much higher proliferative capacity than primary cell lines do, which allows for research to be conducted more efficiently.

What scientific, math, and/or engineering methods will be applied?

To achieve our goal, we will primarily apply cell culture and genetic engineering methods to grow our BSCs and then modify them according to our aims. Specifically, the mammalian transfection methods will allow a plasmid construct to incorporate a gene into our cells using CRISPR, transposons, or Recombinase Mediated Cassette Exchange. Using these systems will also necessitate the application of bacterial culture techniques. Additionally, we will use sequencing methods to ensure that our genes of interest are being inserted properly, as well as imaging to assess the growth and survival of our edited cells.

What realistic constraints (cost, safety, reliability, aesthetics, ethics and social impact, etc.) are to be considered?

What alternative solutions or changes to the plan will be considered?

If we encounter problems while we are creating our cell line, there are many changes we could make to our plan that could improve our results. Instead of just TERT and CDK4, we could try to add other sequences, such as Cyclin D1. We are also already prepared to attempt different genetic engineering methods beyond our first choice of CRISPR, with Sleeping Beauty transposons and Recombinase Mediated Cassette Exchange being our first options.

What are the planned tests and what are the quantitative milestones that will demonstrate achievement of the objectives?

To test successful transfection of our genes, we will sequence our BSCs and look for them, where achievement of our objectives would be demonstrated by the confirmed presence of our gene sequences along with sustained viability of our modified cells. Following successful modification, our next test would be observing our altered BSCs and comparing their ability to divide to unaltered BSCs. Quantitative milestones indicating success would be if our cells divided for longer, as measured by cell concentration over time or number of possible passages.

Competition: what else is going on in the field that would compete with the project plans?

Other methods of finding immortal cells to produce cultured meat include working with stem cells, whether natural or induced, or converting other cell types such as fibroblasts into muscle cells post-proliferation. These methods face significant difficulties with cost and efficiency that our approach aims to avoid. There are also other efforts underway to produce a modified immortalized cell line, whether via genetic engineering or small molecule methods, which occupy a similar space to our project.

Immortalization of Bovine Satellite Cells through TERT and CDK4 Transfection

Team: Miles Arnett, Tina Guo, Addison Mirliani

PI: Andrew Stout

Faculty Advisor: David Kaplan

Abstract

There is currently a significant need for alternative sources of meat-based protein, largely because of the environmental burden of factory farming. One promising method of meeting this need is cultured meat, which aims to produce meat products from cultured animal cells. However, the field has been hampered since its inception by problems with experimental cost and scale-up efficiency. 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. This would allow for much less initial investment in purchasing bovine cells for experimental purposes while also making it far easier to generate large numbers of them. We intend to achieve this goal by using CRISPR, transposons, or Recombinase Mediated Cassette Exchange (RMCE) to insert genes of interest into the BSCs, which promote the expression of telomerase reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4). This method has proven effective in the immortalization of other cell types, but has yet to be applied to BSCs. We will quantify our success by sequencing the cells we produce to identify our genes of interest and by assessing how long our cells can divide under in vitro cell culture relative to a control. Successful completion of this aim would be a significant step forward for efforts to produce cultured meat, which would have a strong positive impact on the accessibility, ethical merit, and ecological sustainability of meat consumption.

Key Words: cultured meat, genetic engineering, senescence

Elements of Engineering Design

This project aims to design a genetically-modified cell line that will improve the process of Bovine Satellite Cell culture. Achieving this aim will also involve designing a process for transfecting relatively large immortalization genes into BSCs, which has yet to be done.

Our first objective towards these goals is to construct plasmids containing our genes of interest, TERT and CDK4, and transfect them into BSCs using CRISPR-Cas9 machinery. This is a continuation of the previous endeavor in which our PI attempted to immortalize these cells, and will provide a good baseline level of effectiveness for our ability to modify them. Our next objective will be to image and analyze our altered cells to assess whether or not our transfection was successful, which will inform our next steps.

If the transfection is unsuccessful, our next objective will be to assess another gene editing system, likely the Sleeping Beauty transposon method, which is generally better at inserting larger plasmids than CRISPR, at the cost of some control. We will also attempt to use Recombinase Mediated Cassette Exchange, another gene editing system, to give ourselves as many alternative routes to success as possible. When transfection has been achieved, our next objective will be to monitor the proliferation and survival of the edited cells in comparison with a control over a long period of time, as this is ultimately how we will determine if they have been immortalized. We will also likely sequence the edited cells and conduct protein quantification to detect our genes and proteins of interest. Success will be determined by tracking proliferation rate and cell lifespan and by confirming a significant difference in the presence of our genes and proteins of interest above the control.

We will primarily apply cell culture and genetic engineering methods to grow our BSCs and then modify them according to our aims. Specifically, the mammalian transfection methods will allow a plasmid construct to incorporate a gene into our cells using CRISPR, transposons, or Recombinase Mediated Cassette Exchange. Using these systems will also necessitate the application of bacterial culture techniques. Additionally, we will use sequencing methods to ensure that our genes of interest are being inserted properly, as well as imaging to assess the growth and survival of our edited cells.

Our primary constraint is in our gene editing methods, because our plasmid will need to be quite large (~15 kb) due to the size of the genes we are inserting and the homology regions that are required to support transfection.We also need to consider the cost of ordering constructs and the cost of our chosen transfection method, as well as reliability, as our cell line needs to consistently avoid senescence reliably in order to be a useful product for scientists. Our cells also need to survive under typical cell culture conditions, or this product will not be useful. If we encounter problems while we are creating our cell line, a potential alternative solution would be to try other sequences, such as Cyclin D1 instead of just TERT and CDK4. We are also already prepared to attempt different genetic engineering methods beyond our first choice of CRISPR, with Sleeping Beauty transposons and Recombinase Mediated Cassette Exchange being our first options.

Design Flow Chart

Figure 1: A design flowchart for the project thus far and in the immediate future.

The above flowchart outlines the design of this project to this point. Our process began with a thorough literature review, in which we identified what would become our primary gene targets, and the ordering of suitable constructs needed to transfect them. We then moved on to our first experiment, in which we are assessing the ability of the CRISPR-based method to successfully modify our cells. This experiment has thus far consisted of primer design, plasmid creation in E. coli, extraction of our plasmids, and transfection into BSCs, and will now continue with selection for successfully modified cells using the antibiotic G418 and cell imaging methods. Our next step will be to progress to experiment two, in which we will essentially repeat the same process with the Sleeping Beauty transposon system (with some modifications to account for differences in protocol). Once experiment 2 is underway, we will move on to testing RMCE as a third possible transfection method.

Introduction and Background

Cultured meat is a growing field of tissue engineering which aims to create meat products produced from cultured animal cells, rather than the factory farming of livestock. Cultured meat was created from the need to change the numerous negative effects that conventional meat production has on the environment, human health, and animal welfare (Post et al., 2020). Although cultured meat is still in the early stages of development, it shows the potential to change the way we consume meat and may offer consumers a more sustainable option.

However, the cultured meat products currently on the market are too expensive to successfully replace traditional meat in the home and at restaurants, largely because the methods currently used to generate them are inefficient when applied to large-scale production. Thus, in order to create a cultured meat product that is inexpensive and sustainable, it is necessary to make the production of cultured meat a more scalable process (Stephens et al., 2018). This has been a significant challenge to the field since its inception, which has only grown more noticeable as other aspects of cellular agriculture have seen significant advancement. Additionally, the current in vitro meat production paradigm relies on a constant supply of primary satellite cells from cows. Although satellite cells can be extracted from the animals without harming them, the frequency of cell extraction and questionable methods of extraction poses an ethical issue, and detracts from the ecological goals that have partially inspired the development of cultured meat to begin with.

One of the potential solutions to this issue of scalability is to prevent senescence of the cells being grown into cultured meat. Senescence is a mechanism enacted by cells as a defense against potential deterioration, in which they stop dividing and halt cell growth. The process of senescence can be triggered by a variety of different events, though it is most often caused by damage to the cell or age of the cell. As a cell ages, its telomeres become progressively smaller, as every time a cell divides, the lengths of its telomeres decrease. These increasingly-shortening telomeres indicate the age of a cell, and cells with shorter telomeres will often senesce (McHugh et al., 2018).

Senescence can be prevented through the immortalization of primary cells, in our case Bovine Satellite Cells, into a perpetually dividing cell line. One of the ways this can be done is through the addition of a telomerase reverse transcriptase (TERT) gene, which produces an enzyme that stops telomere shortening. This prevents the cell from senescing due to age, as with the check of telomere shortening removed, the cell no longer has a limit on its theoretical number of divisions. However, there are other causes of cell senescence and for this reason, the addition of TERT alone is unlikely to produce a cell line that is immortal under practical conditions. Thus, other ways of combat senescence are needed, one of these being the addition of CDK4. This has been shown to decrease senescence by preventing the stalling of a cell before it reaches the S-phase of the cell cycle. This increases a cell’s ability to divide under potentially adverse circumstances, bolstering its chances of remaining potent after many generations in culture (Ruas et al., 2007). We plan to use both of these methods for the immortalization of our cell line.

Integrated Figure

Figure 2: An integrated figure representing the entire project as it stands

Specific Aims

Specific Aim 1: Evaluate the capacity of CRISPR-Cas9, the Sleeping Beauty transposon system, and RMCE to successfully transfect TERT and CDK4 into BSCs. Our hypothesis is that between the high reliability of CRISPR and the size flexibility of the Sleeping Beauty system and RMCE, at least one of these methods should be able to insert our chosen genes into the BSC genome. Completion of this aim will enable us to assess the effect of the transfected genes and provide a guideline for future researchers working with similar plasmids.

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. Our hypothesis is that these genes, which have been able to functionally immortalize other cell types, will also be effective in BSCs. Successful completion of this aim will either result in an immortal BSC line which can be used in cultured meat research or it will inform future researchers that other genes should be tried in any further immortalization efforts.

Methods

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.

Transfection

The cells were transfected using the reagent Lipofectamine™ 3000. 7.5 uL 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.

Next Steps: Sleeping Beauty

If the previous methods prove unsuccessful, we will explore a Sleeping Beauty transposon method for immortalization. This will be about a 24-day process. We will design primers using Benchling that allow us to transfect at the right location. We have the DNA sequence of our cells already, so we can design primers based off of this sequence that will bind to our DNA at the desired locations. We will then perform PCR using these primers and confirm the success of this process through gel electrophoresis. We will purify the resulting PCR fragments, and use them to perform a Gibson Reaction. We will then transform and sequence E. coli using these fragments. After this is complete, we can follow the same steps we did previously by culturing the E. coli in LB broth, performing a miniprep on the resulting E. coli, and using the resulting purified DNA to transfect Bovine Satellite Cells in culture.

Next Steps: Recombinase-Mediated Cassette Exchange

The “tag and exchange” strategy of recombinase mediated cassette exchange, or RMCE, may also be explored if initial transfection experimentations fall short. Studies show that the bovine Rosa26 locus, or bRosa26, has shown potential as a safe harbor for exogenous DNA sequences, and thus is amenable to genetic engineering methods. Combining RMCE and bRosa26 will theoretically result in higher stability and greater expression while avoiding random integration. First, the bRosa26 region will be tagged with two heterospecific recombination target sites, or RTs. Next, the targeted area between the RTs will be exchanged for the desired gene cassette(our genes of interest) using recombinase. By using this technique, we will be able to track TERT and CDK4 in the BSCs.

Results

Our project does not have a large number of results to display yet, but we are in the process of periodically imaging our cells to track their growth and division post-transfection.

Figure 2: Brightfield images of BSCs 3 days post-transfection using the CRISPR-Cas9 system, with 3 different sgRNAs being tested. Scale bars are 100um.

Figure 3: Brightfield images of BSCs 5 days post-transfection using the CRISPR-Cas9 system, with 3 different sgRNAs being tested. Scale bars are 100um.

Figure 4: Brightfield images of BSCs 7 days post-transfection using the CRISPR-Cas9 system, with 3 different sgRNAs being tested. Scale bars are 100um.

Figure 5: Brightfield images of BSCs 10 days post-transfection using the CRISPR-Cas9 system, with 3 different sgRNAs being tested. Scale bars are 100um.

From these images, it appears as though the control cells are dying faster than any of our modified cells, but it is still too early to say with confidence that this means our transfection was successful. G418 is a relatively slow-acting antibiotic, so we will not be able to determine whether our cells have been made immune to it until more time has passed.

Discussion and Future Work

As discussed previously, aside from attempting more transfection methods, future work at the moment mainly involves testing successful transfection of our genes. To do this, we will sequence our BSCs and look for them, where achievement of our objectives would be demonstrated by the confirmed presence of our gene sequences along with sustained viability of our modified cells. We will also use protein analytical methods to ensure our transfected genes are being transcribed and translated. Following successful modification, our next test would be observing our altered BSCs and comparing their ability to divide to unaltered BSCs. Quantitative milestones indicating success would be if our cells divided for longer, as measured by cell concentration over time or number of possible passages.

Conclusions

Individual Contributions

Each of us has thus far contributed equally to the lab work. Miles has been largely responsible for writing about the specifics of the methods we will be using, Addy about the background, and Tina about the overall project structure.

Acknowledgements

We would like to acknowledge our PI, Andrew Stout, for being extremely helpful throughout this project so far, taking the time to explain how he has been conducting his research to this point and the best way for us to conduct ours. We would also like to acknowledge John Yuen for assisting us while Andrew was away on his honeymoon. Finally, we would like to acknowledge David Kaplan for providing us with lab space and overseeing the BME7 class in general.

References

McHugh, D., & Gil, J. (2018). Senescence and aging: Causes, consequences, and therapeutic avenues. The Journal of cell biology, 217(1), 65–77. https://doi.org/10.1083/jcb.201708092

Post, M. J., Levenberg, S., Kaplan, D. L., Genovese, N., Fu, J., Bryant, C. J., . . . Moutsatsou, P. (2020). Scientific, sustainability and regulatory challenges of cultured meat. Nature Food, 1(7), 403-415. doi:10.1038/s43016-020-0112-z

Ruas, M., Gregory, F., Jones, R., Poolman, R., Starborg, M., Rowe, J., Brookes, S., & Peters, G. (2007). CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms. Molecular and cellular biology, 27(12), 4273–4282. https://doi.org/10.1128/MCB.02286-06

Stephens, N., Di Silvio, L., Dunsford, I., Ellis, M., Glencross, A., & Sexton, A. (2018). Bringing cultured meat to market: Technical, socio-political, and regulatory challenges in cellular agriculture. Trends in Food Science & Technology, 78, 155-166.

doi:https://doi.org/10.1016/j.tifs.2018.04.010

Turan, S., Galla, M., Ernst, E., Qiao, J., Voelkel, C., Schiedlmeier, B., Zehe, C., & Bode, J. (2011). Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. Journal of molecular biology, 407(2), 193–221. https://doi.org/10.1016/j.jmb.2011.01.004