Part:BBa_K4869003
Engineered HEK293 cells producing Afamin and wnt3a proteins
Application in Our Project:
">Figure: Concept model of engineered HEK293 cells producing Afamin and wnt3a proteins.
Our first goal is to establish Afamin-Wnt3a producer cells in vitro. We will do this by transfecting cells with a plasmid containing the Afamin-Wnt3a gene. Once the cells successfully produce Afamin-Wnt3a protein, we will use them to generate conditioned media for our downstream experiments.
Transfection is the process of introducing foreign DNA or RNA into cells. In this case, we want to introduce a plasmid containing the Afamin-Wnt3a gene into HEK293 cells. Once the plasmid is inside the cell, it can be transcribed and translated into Afamin-Wnt3a protein. This plasmid can be transfected into HEK293 cells, which are a type of human embryonic kidney cell line that is commonly used in scientific research.
When the Afamin-Wnt3a gene is expressed in these cells, it leads to the production of the Afamin-Wnt3a protein. One way to introduce the Afamin-Wnt3a Plasmid into HEK293 cells is through the Super piggyBac Transposon vector. Super piggyBac Transposon vector contains the PiggyBac transposase gene, an enzyme that can integrate the Afamin-Wnt3a Plasmid into the genome of the HEK293 cells. Once integrated, the Afamin-Wnt3a gene sequence becomes a part of the cell's DNA and is passed on to daughter cells during cell division.
By using the Super piggyBac Transposon vector to generate Afamin-Wnt3a producer cells, we can produce large quantities of Afamin-Wnt3a protein for use in downstream experiments. With a combination of those following genomes, the plasmid containing the Afamin-Wnt3a gene must be transfected to the genome of HEK293 cells to establish Afamin-Wnt3a producer cells. HEK293 cells are also known as human embryonic kidney cells, which are robust and fast-growing and, therefore, have been frequently used for receptor signaling, cancer research, and large-scale protein production.
To successfully integrate the foreign genes into the host genome, one strategy is to utilize the Super piggyBac Transposon vector. Super piggyBac transposase gene inside the transposon vector is the enzyme playing the crucial role of transfecting the plasmid into the HEK293 cells genome. Once transfected, the Afamin-Wnt3a gene will be integrated within the HEK293 cells’ DNA and passed on to other cells during cell division. Consequently, Afamin-Wnt3a producer cells may be generated utilizing the Super piggyBac Transposon vector.
We utilized the engineered HEK293 cells by designing the cells, using Super piggyBac transposase expression vector and BII-CMV-AfmW3A vector, to produce Afamin Wnt3a protein complex enriched conditioned media. To determine the optimal conditions for the transfection process, we conducted experiments with two different plasmid vector molar concentration ratios between the BII-CMV-AfmW3A vector and Super PiggyBac Transposase Expression Vector. After the Afamin Wnt3a protein complex, which is initially made in the cytosol of the cell, is secreted into the cell media by the HEK293 cell, we collected the enriched conditioned media, which is a replacement for FBS.
References:
Coulter, B. (n.d.). An overview of HEK-293 cell line. Beckman Coulter Life Sciences. www.beckman.kr/resources/product-applications/lead-optimization/cell-line-development/human-embryonic-kidney-293#:~:text=Human%20Embryonic%20Kidney%20(HEK)%20293,for%20their%20propensity%20for%20transfection.
Grainger, S., & Willert, K. (2018, September). Mechanisms of Wnt Signaling and control. Wiley interdisciplinary reviews. Systems biology and medicine. www.ncbi.nlm.nih.gov/pmc/articles/PMC6165711/.
Synthego. (n.d.). Full stack genome engineering. Synthego. www.synthego.com/hek293#:~:text=HEK293T%20cells%20are%20a%20popular,commonly%20used%20for%20retroviral%20production.
Sequence and Features
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