- Email: [email protected]
Part V. Modulation of Gene Expression
Part V
Modulation of Gene Expression Congratulations! You have made tremendous headway in learning and successfully manipulating recombinant DNA. Thus far, you have created a recombinant pET-41a expression vector containing the egfp gene and transformed this plasmid into the model organism E. coli to express and purify the GST::EGFP protein. You may have also induced the overexpression of the protein in E. coli using IPTG, purified the protein, as well as quantified the amount of gene expression by using RT-qPCR to examine gst::egfp mRNA levels. The goal of the following advanced series of experiments is to take your scientific skills to the next level. You will transfect (introduce) the egfp gene in a different in vitro model system, HEK293 human embryonic kidney cells, a mammalian cell culture model. Once you verify through fluorescence screening that HEK293 cells express the EGFP protein, instead of upregulating (overexpressing) egfp as you did in E. coli you will now downregulate expression of the egfp gene. To do this, you will utilize two cutting-edge and effective techniques: RNA interference (RNAi) and CRISPR-Cas9. Each method has its own unique advantages and disadvantages, which are explained in the introductions of the respective lab sessions. In the next series of lab sessions, you will create a stable HEK293 cell line that constitutively (always) expresses the EGFP protein using the mammalian expression vector pEGFP-N1. Next, you will screen HEK293 cells transfected with the pEGFP-N1 plasmid by fluorescence microscopy to determine successful transfection of the egfp transgene. Finally, you will downregulate the expression of the EGFP protein in HEK293 cells by designing siRNA molecules to specifically target and knockdown the expression of egfp mRNA, thus considerably reducing the amount of EGFP protein. Finally, you will downregulate expression of egfp by designing guide RNAs to be used in the CRISPR-Cas9 system and compare and contrast your results. All the experiments from this point forward will require you to have a good understanding and knowledge of how to work with mammalian cells in culture. Therefore, mastering your aseptic technique is a crucial first step.
TIME LINE The experiments proposed in Part V, Modulation of Gene Expression are designed to be completed as a complement to Parts I IV, conducted during the following 15-week semester. However, due to the complexity of experiments, we suggest those with time limitations commercially purchase HEK293 cells expressing GFP (Note: not EGFP) in lieu of creating them in Lab Session 19, Transient Transfection of Mammalian Cells (Fig. V.1). These cells are available from Cell BioLabs (Catalog #: AKR-200). Furthermore, these labs are designed to be flexible to fit various curricular needs. Therefore, it may be suitable for students to only create stable cells expressing EGFP (Lab Sessions 18 19), while other student learning outcomes may require students to experiment further to knockdown and/or knockout gene expression utilizing RNAi (Lab Sessions 20 22) or the CRISPR-Cas9 system (Lab Sessions 23 30), respectively. Please refer to Fig. V.1 to choose the path of experiments that is most suitable for your course. 135
136
PART | V Modulation of Gene Expression
FIGURE V.1 Proposed time line for experiments outlined in Part V. If stable HEK293 cells expressing GFP are purchased commercially, students will begin with Lab Session 18, Culturing Mammalian Cells and skip Lab Session 19, Transient Transfection of Mammalian Cells. Note: Lab Session 19c “Creating a Kill Curve” takes approximately two weeks to complete and needs to be finished by the beginning of the course.
Modulation of Gene Expression Congratulations! You have made tremendous headway in learning and successfully manipulating recombinant DNA. Thus far, you have created a recombinant pET-41a expression vector containing the egfp gene and transformed this plasmid into the model organism E. coli to express and purify the GST::EGFP protein. You may have also induced the overexpression of the protein in E. coli using IPTG, purified the protein, as well as quantified the amount of gene expression by using RT-qPCR to examine gst::egfp mRNA levels. The goal of the following advanced series of experiments is to take your scientific skills to the next level. You will transfect (introduce) the egfp gene in a different in vitro model system, HEK293 human embryonic kidney cells, a mammalian cell culture model. Once you verify through fluorescence screening that HEK293 cells express the EGFP protein, instead of upregulating (overexpressing) egfp as you did in E. coli you will now downregulate expression of the egfp gene. To do this, you will utilize two cutting-edge and effective techniques: RNA interference (RNAi) and CRISPR-Cas9. Each method has its own unique advantages and disadvantages, which are explained in the introductions of the respective lab sessions. In the next series of lab sessions, you will create a stable HEK293 cell line that constitutively (always) expresses the EGFP protein using the mammalian expression vector pEGFP-N1. Next, you will screen HEK293 cells transfected with the pEGFP-N1 plasmid by fluorescence microscopy to determine successful transfection of the egfp transgene. Finally, you will downregulate the expression of the EGFP protein in HEK293 cells by designing siRNA molecules to specifically target and knockdown the expression of egfp mRNA, thus considerably reducing the amount of EGFP protein. Finally, you will downregulate expression of egfp by designing guide RNAs to be used in the CRISPR-Cas9 system and compare and contrast your results. All the experiments from this point forward will require you to have a good understanding and knowledge of how to work with mammalian cells in culture. Therefore, mastering your aseptic technique is a crucial first step.
TIME LINE The experiments proposed in Part V, Modulation of Gene Expression are designed to be completed as a complement to Parts I IV, conducted during the following 15-week semester. However, due to the complexity of experiments, we suggest those with time limitations commercially purchase HEK293 cells expressing GFP (Note: not EGFP) in lieu of creating them in Lab Session 19, Transient Transfection of Mammalian Cells (Fig. V.1). These cells are available from Cell BioLabs (Catalog #: AKR-200). Furthermore, these labs are designed to be flexible to fit various curricular needs. Therefore, it may be suitable for students to only create stable cells expressing EGFP (Lab Sessions 18 19), while other student learning outcomes may require students to experiment further to knockdown and/or knockout gene expression utilizing RNAi (Lab Sessions 20 22) or the CRISPR-Cas9 system (Lab Sessions 23 30), respectively. Please refer to Fig. V.1 to choose the path of experiments that is most suitable for your course. 135
136
PART | V Modulation of Gene Expression
FIGURE V.1 Proposed time line for experiments outlined in Part V. If stable HEK293 cells expressing GFP are purchased commercially, students will begin with Lab Session 18, Culturing Mammalian Cells and skip Lab Session 19, Transient Transfection of Mammalian Cells. Note: Lab Session 19c “Creating a Kill Curve” takes approximately two weeks to complete and needs to be finished by the beginning of the course.