Students will contribute to research using CRISPR technology to create complete gene knockout lines, helping to clarify the exact function of the gene Apol11b.
This project has reached full capacity for the current term. Please check back next semester for updates.
We have previously determined that the gene Apol11b is transiently expressed during the process of erythroid terminal differentiation. However, the exact function of this gene remains unknown. Recent experiments using interfering RNA suggest it may play a role in hemoglobin production, as in cells carrying a silencing construct there is a clear differences in hemoglobin accumulation compared to controls. The data concerning expression of Apol11b mRNA and protein have been inconsistent, possibly due to variability in the silencing of the gene by siRNA. We now will take the approach of creating complete gene knockout lines using CRISPR technology to generate more consistent and reproducible results.
Students will be expected to keep a detailed digital laboratory notebook on Dr. Koury's UB Box drive and present their results at regular lab meetings held with Dr. Koury and other graduate/undergraduate students in the lab. Data generated of sufficient quality will also be included in manuscripts/presentations at meetings with co-authorship for the student researchers. Data will also be presented at UB and possibly other regional student-centered scientific meetings in poster format in the spring semester.
|Length of commitment||About a semester (3-5 months)|
|In-person, remote, or hybrid? ||In-person|
|Level of collaboration||Small group project (2-3 students)|
|Who is eligible||Students with good basic laboratory skills, familiarity with basic laboratory instrumentation, pipetting, some cell culture and record keeping, hands-on experience with basic molecular biology techniques desired|
This image shows results driving the development of this project. It shows pellets of murine erythroleukemia cells in their uninduced state ( A and B) and after being induced to undergo a limited erythroid differentiation for 3 days in the presence of DMSO (C and D). Pellets A and D are from cells transducer with a lentivirus that expressions a "scrambled" small inferring RNA (control siRNA). As these cells undergo induction, hemoglobin accumulates in their cytoplasm, and this is why the pellet appears red in C. In contrast, cells in pellets B and D have been transducer with lentivirus expression an siRNA specific for Apol11b mRNA ( silencing siRNA). This siRNA should "knockdown" expression of Apol11b so that we can determine what effect reducing expression of this gene has on the cells. In this image it can be seen that the cells expressing the silencing siRNA are markedly less red...even in the unindexed state, suggesting Apol11b might have a role in the synthesis of heme.
This figure shows an example of results from quantitative real time PCR measurement of Apol11b mRNA levels in MEL cells, MEL cells transduced with the control siRNA (Control) and in MEL cells transduced with Apol11b siRNA (Apol11b). The values are the mean fold change values for the 3 cell types calculated over 3 induction sets and plotted against the individual cell types. Significant difference in fold changes between MEL and ApoL11b knockout cells was observed in this experiment, but not between Apol11b knockdown and control MEL cells. Further, there appeared to be a decreased expression of Apol11b in the control cells compared to the wild type MEL cells, suggesting a non-specific effect of lentiviral transduction on expression of Apol11b . Other experiments have been even less clear, prompting us to consider the use of the CRISPR-Cas system to completely knockout the Apol11b gene to get a more specific indicator of the function of this gene.
This image shows the three plasmid constructs that will be purchased for this project. Once will simultaneously express the guide RNA and Cas9 enzyme that will cut the muring Apol11b gene in the MEL cells (MCP273405-CG12-3), while the plasmid containing the donor clone will insert an antibiotic resistance gene into the cut Apo11b gene to completely knock it out and allow for selection of MEL cells with the knockout. The third plasmid will be used to generate a control transfected MEL cell line to use for comparison in the study.
Once you begin the digital badge series, you will have access to all the necessary activities and instructions. Your mentor has indicated they would like you to also complete the specific preparation activities below. Please reference this when you get to Step 2 of the Preparation Phase.
Biotechnical and Clinical Laboratory Sciences