Teodora G Georgieva

Interests

Research

Design and construction of gene targeting and transgenic vectors, gene editing using CRISPR/Cas9.

Courses

Scholarly Contributions

Journals/Publications

• Christine, B. M., Teodora, G., Trevor, T. N., Tama, T., Larry, J., Jennifer, U. L., David, B., & Janko, N. Ž. (2024). Cutting edge: Characterization of low copy number human angiotensin-converting enzyme 2 (hACE2)-transgenic mice as an improved model of SARS-CoV-2 infection.. J Immunology.
• Georgieva, T. G., Lee, N. Y., Mythreye, K., Langlais, P. R., Hund, T. J., Mohler, P. J., Largent-Milnes, T., Vanderah, T. W., Lee, Y. S., Mouneimne, G., Ellis, N. A., Lochhead, J. J., Ortiz, H. R., Ahmed, T., Cruz-Flores, P., Kumar, S., Ramonett, A., Pan, C. C., & Kwak, E. (2022). βIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature Communications. doi:10.1038/s41467-022-28933-1
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  Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report βIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific βIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, βIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, βIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.
• Mustacich, D. M., Kylathu, R., Bernas, M. J., Myles, R. J., Jones, J. A., Kanady, J. D., Simon, A., Georgieva, T. G., Witte, M. H., Erickson, R. P., & Pires, P. (2021). Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin47 R260C point mutation.. Lymphology, 54(2), 78-91.
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  Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p
• Pires, P., Erickson, R. P., Witte, M. H., Georgieva, T. G., Simon, A., Kanady, J. D., Jones, J. A., Myles, R. J., Bernas, M. J., Kylathu, R., & Mustacich, D. M. (2021). Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin 47 R260C point mutation. Lymphology, 54(1), 78-91.
• Pires, P., Erickson, R., Witte, M., Georgieva, T., Simon, A., Kanady, J., Jones, J., Mustacich, D., Kylat, R., Bernas, M., & Myles, R. (2021). ABNORMAL LYMPHATIC PHENOTYPE IN A CRISPR MOUSE MODEL OF THE HUMAN LYMPHEDEMA-CAUSING CONNEXIN47 R260C POINT MUTATION. Lymphology, 54(2). doi:10.2458/lymph.4729
• Powell, D. A., Hsu, A. P., Shubitz, L. F., Butkiewicz, C. D., Moale, H., Trinh, H. T., Doetschman, T., Georgieva, T. G., Reinhartz, D., Wilson, J., Orbach, M. J., Holland, S. M., Galgiani, J. N., & Frelinger, J. A. (2022). "Mouse model of a human STAT4 point mutation that predisposes to disseminated Coccidiomycosis (DCM)". ImmunoHorizons, 6(2), 116-129. doi:https://doi.org/10.4049/immunohorizons.2000072
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  STAT4 plays a critical role in the generation of both adaptive and innate immune responses. In the absence of STAT4, Th1 responses do not occur which are critical for resistance to fungal disease. Infection with the dimorphic fungus, Coccidioides is a major cause of community acquired pneumonia in the endemic regions of Arizona and California. In some people, and often for unknown reasons, infection with Coccidioides results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis. Surprisingly all affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/+). A knock-in mouse, heterozygous for the substitution, was deficient in production of IFN-gamma after anti-CD3/CD28 stimulation. As with the human family, mice heterozygous for the mutation were equally more susceptible to experimental coccidioidal infection than WT mice. As expected, mice lacking STAT4 were also more susceptible. Together these observations are consistent with STAT4E626G functioning as a dominant negative STAT4 allele. Supporting this, spleen cells from STAT4E626G mice show defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early after infection, mutant B6-STAT4E626G/+ mice failed either to produce pulmonary IFN-gamma and related cytokines or to accumulate activated adaptive immune cells in mediastinal lymph nodes, consistent with blunted STAT4 signaling. These results point to a deficiency in the innate host immune response can lead to progressive coccidioidomycosis.
• Napierski, N. C., Granger, K., Langlais, P. R., Moran, H. R., Strom, J., Touma, K., & Harris, S. P. (2020). A Novel "Cut and Paste" Method for In Situ Replacement of cMyBP-C Reveals a New Role for cMyBP-C in the Regulation of Contractile Oscillations. Circulation research, 126(6), 737-749.
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  cMyBP-C (cardiac myosin-binding protein-C) is a critical regulator of heart contraction, but the mechanisms by which cMyBP-C affects actin and myosin are only partly understood. A primary obstacle is that cMyBP-C localization on thick filaments may be a key factor defining its interactions, but most in vitro studies cannot duplicate the unique spatial arrangement of cMyBP-C within the sarcomere.
• Georgieva, T., & Doetschman, T. (2017). Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease. Circulation Research, 120(5), 876-894. doi:10.1161/circresaha.116.309727
• Georgieva, T., Dunkov, B. C., Harizanova, N., Ralchev, K., & Law, J. H. (1999). Iron availability dramatically alters the distribution of ferritin subunit messages in Drosophila melanogaster. Proceedings of the National Academy of Sciences, 96(6), 2716-2721. doi:10.1073/pnas.96.6.2716

Poster Presentations

• Georgieva, T. G. (2021, April 21). Genetically Engineered Mouse Models (GEMM) EMSR Unit – Supporting your research by creating custom mouse models. University of Arizona Cancer Center Cancer Biology Program retreat. Online, zoom: UACC.
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  EMSR’s GEMM unit is a well-established part of the shared resource. We provide a wide range of “sequence-to-whiskers” services: transgenic, gene targeted and CRISPR-edited mouse models, sperm cryo preservation and storage, IVF and embryo rederivation, free in-depth consultation, vector and genotyping troubleshooting. With the start of the COVID 19 pandemic our core with a financial aid from the BIO5 Institute, was able to react fast in an effort to complement the SARS2 and COVID research of UA scientists with the generation of transgenic mice expressing the hACE2 receptor under the regulation of keratin 18 promoter. We received the transgenic vector by the courtesy of its original creators (P.B. McGray et al, J. Virology, 2006) and microinjected it into Bl6 NJ embryos to produce 7 founders carrying the transgene. We have determined gene copy number by quantitate PCR and the expression levels of hACE2 receptor in the lungs of our founders. Based on the results we have decided to continue and breed two of those founders to establish colonies. One of our current colonies carries one copy of the transgene and the other has two copies, in comparison with the 8 transgene copies in the original mice distributed by JAX Lab. The transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease in the mice with 8 copies, so we expect that the mice developed by us will give the investigators more time to study the progression of the disease upon virus exposure. Currently these COVID mice are available for distribution to the interested researchers.
• Georgieva, T. G., Doetschman, T., Johnson, L., & Taylor-Doyle, T. (2021, March 30- April 7, 2021). Genetically Engineered Mouse Models (GEMM) Core Facility – Supporting your research by creating “Sequence to Whiskers” custom mouse models. Discover BIO5 Research to Innovation Showcase. BIO5 Institute, University of Arizona.
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  GEMM Core is a well-established University core facility. We provide a wide range of “sequence-to-whiskers” services: transgenic, gene targeted and CRISPR-edited mouse models, sperm cryo preservation and storage, IVF and embryo rederivation, free in-depth consultation, vector and genotyping troubleshooting. For the 13 years of our existence we have made 173 models: 47 transgenic, 54 gene-targeted and 72 CRISPR-edited mouse models. Presently, GEMM Core employs the cutting-edge technology of Cas9/CRISPR genome editing as a routine method for generation of simple and conditional knockouts, domain deletions/substitutions, small and fluorescent tag knock-ins, sensor-sensitive tag-knockins and SNPs. In the last year we significantly expanded the capabilities of CRISPR-based conditional knockout gene targeted models through the application of long single stranded DNA as a template for CRISPR homology-directed repair. As this service costs significantly less than traditional gene targeting services we hope to better position UA research.
• Georgieva, T. G., Doetschman, T., Johnson, L., & Taylor-Doyle, T. (2021, November 20th). Genetically Engineered Mouse Models (GEMM) Core Facility – Supporting your research by creating custom mouse models. University of Arizona Cancer Center Scientific Retreat. HSIB, UA: University of Arizona Cancer Center.
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  GEMM Core is a well-established University core facility. We provide a wide range of “sequence-to-whiskers” services: transgenic, gene targeted and CRISPR-edited mouse models, sperm cryo preservation and storage, IVF and embryo rederivation, free in-depth consultation, vector and genotyping troubleshooting. For the 13 years of our existence we have made 160 models: 45 transgenic, 54 gene-targeted and 61 CRISPR-edited mice with approximately 25% of them for members of UACC. Presently, GEMM Core employs the cutting-edge technology of Cas9/CRISPR genome editing as a routine method for generation of simple and conditional knockouts, domain deletions/substitutions, small and fluorescent tag knock-ins, sensor-sensitive tag-knockins and SNPs. In the last year we significantly expanded the capabilities of CRISPR-based conditional knockout gene targeted models through the application of long single stranded DNA as a template for CRISPR homology-directed repair. As this service costs significantly less than traditional gene targeting services and by taking advantage of the 50/50 matching fund we hope to better position UACC members for cancer research.
• Georgieva, T. G., Doetschman, T., Johnson, L., & Taylor-Doyle, T. (2021, October 26-29, 2020). University of Arizona Genetically Engineered Mouse Models (GEMM) Core Facility – Supporting research by creating custom mouse models. TT2020 - 16TH TRANSGENIC TECHNOLOGY MEETING. Virtual: The Weizmann Institute, Rehovot, Israel.
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  GEMM Core is a well-established University of Arizona core facility. We provide a wide range of “sequence-to-whiskers” services: transgenic, gene targeted and CRISPR-edited mouse models, sperm cryo preservation and storage, IVF and embryo rederivation, free in-depth consultation, vector and genotyping troubleshooting. For the 15 years of our existence we have made 181 models: 47 transgenic, 54 gene-targeted and 80 CRISPR-edited mice. Presently, GEMM Core employs the cutting-edge technology of Cas9/CRISPR genome editing as a routine method for generation of simple and conditional knockouts, domain deletions/substitutions, small and fluorescent tag knock-ins, sensor-sensitive tag-knockins and SNPs. In the last year we significantly expanded the capabilities of CRISPR-based conditional knockout gene targeted models through the application of long single stranded DNA as a template for CRISPR homology-directed repair. Some of these models have been created to study predisposition of disseminated Coccidiomycosis (DSM), known as Valley Fever (Journal of Immunology, submitted) or the identification of Mfn2-S249 as a Phosphoregulatory Switch of Mitochondrial Fusion Dynamics (Nature Comm, in press). With the start of the COVID 19 pandemic we have generated transgenic mice expressing the hACE2 receptor under the regulation of keratin 18 promoter to complement the SARS2 and COVID research of UA scientists. The transgenic mice were characterized by determining copy numbers, lung tissue expression level and neutralization titers.

Others

• Georgieva, T. G. (2021, June). New CRISPR/Cas technologies. 11th Workshop Innovative Mouse Models.