1
Abstract Using CRISPR Knockout of Tie2 Gene to Study Apoptotic Resistance in Human Embryonic Stem Cells Tie2 receptor in the Angiopoietin 1 signaling pathway plays a vital role in preventing apoptosis from irradiation in human embryonic stem cells (hESCs). The same pathway is present in cancer cells, allowing the cells to survive chemotherapy treatment, because of signals from dying daughter cells to the Tie2 receptor. Having the ability to inhibit the receptor can lead to having the cells be affected by the chemotoxic agents, and being able to kill the cells. In order to learn more about the Angiopoietin 1 signaling pathway in human embryonic stem cells, we will inhibit the receptor by creating a Tie2 naïve knockout of cell line. This cell line will have Tie2 gene deleted entirely from the cell’s genome. We used CRISPR genome editing technique to knockout Tie2 gene in hESCs. The results of this method have not yet been gathered; however, more knowledge on the Angiopoietin 1 pathway will potentially be used in making cancer treatment by irradiation more effective. Arlene Rivas Barrios 1 , Amy Ferreccio 2 , Brock William Hashim 3 , Sonia Beant Sidhu 2 , Hannele Ruohola-Baker 2 1 UW GenOM Project, 2 Department of Biochemistry, University of Washington, Seattle, Washington , 3 Eastern Virginia Medical School Methods Results Acknowledgments References • Brindle, Nicholas P.J. Signaling and Functions of Angiopoietin-1 in Vascular Protection. Digital image. Circulation Research. American Heart Association, 2015. Web. 16 Aug. 2015. • In Vitro Transcription: In Vitro Transcription. Digital image. Cambio, n.d. Web. 16 Aug. 2015. • CRISPR-P, “A Web Tool For Synthetic Single-Guide RNA Design of CRISPR-System In Plants.”(2015). Web. 10 Aug 2015. • Federico Gonzalez, et al. “An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells.” Developmental Biology Program, Sloan-Kettering Institute. (2014). Web. 10 Aug. 2015. • Post Chemotherapy Stem Cells Treatment For Cancer. Digital image. Customized Treatment on Cancer by Cellular Medicine. StemRX, n.d. Web. 16 Aug. 2015. • Xing Yalan, Su TT, and Ruohola-Baker Hannele. "Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.“(2015). Web. 1 Aug. 2015. A special thank you to Dr. Hannele Ruohola-Baker for the invitation and the opportunity to work in her lab. Also, a big thank you to Amy Ferreccio for her mentorship and guidance throughout the research. I would like to also acknowledge Brock Hashim and Sonia Beant Sidhu for their help and input on the project. Also, a tremendous thank you to Drs. Anne Dinning and Michael Wolf for their generous gift. Finally, a big thanks to UW GenOM Project (NIH5R25HG007153-03) and NIH funding to Dr. Ruohola-Baker for the great opportunity to learn and participate in an amazing program. In memory of Pink Henrik. University of Washington GenOM Project: ALVA 2015 • We show that this is caused by a “community effect”: apoptotic daughter cells send a signal to protect the matriarch, adult stem cells (Xing et al., 2015) • Pathway that protects drosophila stem cells from apoptosis is homologous to the ang-1 pathway in humans • Tie2 receptor in the ang-1 pathway is believed to be the determinant of stem cell survival • Clustered Regularly Spaced Palindromic Repeats (CRISPR) is an immune response Originally discovered in bacteria that can be used in gene editing and regulation • CRISPR can be used to delete the Tie2 gene from a hESC’s genome. cancer cells to create apoptosis in the cells, eradicating cancer 1. Design guides 2. PCR and gel electrophoresis 3. PCR purification 4. In Vitro transcription 5. Treat cells with doxycycline 6. Transfection 7. DNA extraction 8. PCR to test for mutations and sequencing Figure 1. Resulting gel after PCR of primer sets 1A, 1C, 2A, and 3B. Figure 2. Resulting gel after PCR of primer set 1A. Figure 3. Resulting gel after testing combination of primers, 3 of which worked: 1A and 2A, 2A and 3B, and 2A and 4A. Figure 4. Primers that worked were used to test for mutations by going through PCR with template DNA from ELF hESCs, a combination of guides, and a control cancer DNA. None worked, but the appearance of no band in the template DNA using primers 3B and guides 2,3, and 4 indicated the possibility of a mutation. Figure 5. Another PCR reaction and gel was then performed on the template DNA and control using guides 2,3, and 4. The resulting gel showed no mutations. • After analyzing the gels, it was apparent that the primers and guides did not successfully target and delete the Tie2 gene. • Possible solutions could be to adjust annealing temperatures or re-evaluate the design of the guides and primers. Once a mutation can be attained, the DNA can then be sent for sequencing. • The next step is to perform phenotypic analysis of Tie2 KO in Elf hESCs to elucidate its role in programmed cell death. The fly homolog of Angiopoietin1 pathway prevents programmed cell death in fly stem cells. If the same is true in humans, a CRISPR knockout cell line of the Tie2 will not survive irradiation nor chemotoxic agents when rescued by Angiopoietin1. Ladder 1A 1C 2A 3B Template: +C g12 +C g12 +C g12 +C g12 +C g12 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1A Ladder Non Irradiated Conditioned Media Conditioned media • Aggressive cancers are resistant to apoptosis • The molecular mechanism of this resistance is poorly understood • Stem cells are also resistant to genotoxic agents Hypothesis Introduction
