Diana Ji

Targeted integration in rat and mouse embryos with zinc-finger nucleases

Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell–based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.

Targeted Genome Modification in Mice Using Zinc Finger Nucleases

Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20–75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.

Whole-rat conditional gene knockout via genome editing

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter–driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

The kinase Mirk is a potential therapeutic target in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor affecting children and adolescents. The majority of patients are treated by surgery and chemotherapy but have limited alternative therapeutic options. Kinases play an important role in the growth and survival of tumor cells. We aim to identify specific kinases to be vital in the survival of osteosarcoma cells and thus may be a key target in creating novel anticancer therapies. A lentiviral short hairpin RNA kinase library, screened osteosarcoma cells, identified kinase minibrain-related kinase (Mirk) (Dyrk1B) as a potential target. Knockdown Mirk expression could inhibit cell growth and induce apoptosis. Chemically synthetic small interfering RNA knockdown and complementary DNA rescue assay further confirmed the results from the decrease of Mirk gene expression. The relationship between Mirk gene expression and the clinical characteristics of patients with osteosarcoma was investigated using tissue microarray and immunohistochemistry analysis. The data indicate that the overall survival rate of patients with Mirk high staining (high levels of Mirk protein expression) is significantly shorter than those with Mirk low staining and moderate staining. This highlights Mirks potential to serve as a promising target for molecular therapy in the treatment of osteosarcoma.

Lentiviral shRNA screen of human kinases identifies PLK1 as a potential therapeutic target for osteosarcoma

We describe an optimized systematic screen of known kinases using osteosarcoma cell lines (KHOS and U-2OS) and a lentiviral-based short hairpin RNA (shRNA) human kinase library. CellTiter 96(R)AQueous One Solution Cell Proliferation Assay was used to measure cell growth and survival. We identified several kinases, including human polo-like kinase (PLK1), which inhibit cell growth and induce apoptosis in osteosarcoma cells when knocked down. cDNA rescue and synthetic siRNA assays confirm that the observed phenotypic changes result from the loss of PLK1 gene expression. Furthermore, a small molecule inhibitor to PLK1 inhibited osteosarcoma cell growth and induced apoptosis. Western blot analysis confirmed that PLK1 is highly expressed and activated in several osteosarcoma cell lines as well as in resected tumor samples. Immunohistochemistry analysis showed that patients with high PLK1 tumor expression levels correlated with significantly shorter survival than patients with lower levels of tumor PLK1 expression. These results demonstrate the capability and feasibility of a high-throughput screen with a large collection of lentiviral kinases and its effectiveness in identifying potential drug targets. The development of more potent inhibitors that target PLK1 may open doors to a new range of anti-cancer strategies in osteosarcoma.

Lentiviral short hairpin RNA screen of genes associated with multidrug resistance identifies PRP-4 as a new regulator of chemoresistance in human ovarian cancer

Published reports implicate a variety of mechanisms that may contribute to drug resistance in ovarian cancer. The chief aim of this study is to understand the relationship between overexpression of drug resistance associated genes and multidrug resistance in ovarian cancer. Using lentiviral short hairpin RNA collections targeting 132 genes identified from transcriptional profiling of drug-resistant cancer cell lines, individual knockdown experiments were done in the presence of sublethal doses of paclitaxel. Specific genes whose knockdown was found to be associated with cellular toxicity included MDR1 (ABCB1), survivin, and pre-mRNA processing factor-4 (PRP-4). These genes, when repressed, can reverse paclitaxel resistance in the multidrug-resistant cell line SKOV-3TR and OVCAR8TR. Both MDR1 and survivin have been reported previously to play a role in multidrug resistance and chemotherapy-induced apoptosis; however, the effect of PRP-4 expression on drug sensitivity is currently unrecognized. PRP-4 belongs to the serine/threonine protein kinase family, plays a role in pre-mRNA splicing and cell mitosis, and interacts with CLK1. Northern analysis shows that PRP-4 is overexpressed in several paclitaxel-resistant cell lines and confirms that PRP-4 expression could be significantly repressed by PRP-4 lentiviral short hairpin RNA. Both clonogenic and MTT assays confirm that transcriptional repression of PRP-4 could reverse paclitaxel resistance 5-10-fold in SKOV-3TR. Finally, overexpression of PRP-4 in drug-sensitive cells could induce a modest level of drug resistance to paclitaxel, doxorubicin, and vincristine. [Mol Cancer Ther 2008;7(8):2377–85]

Corrigendum: Whole-rat conditional gene knockout via genome editing

In the version of this article initially published, we described a method to identify tumor-initiating cells from human primary gliomasphere cell cultures or directly from fresh human glioma specimens. We used FACS analysis to describe a tumor-initiating cell subpopulation that displayed a specific morphology (high forward scatter, low side scatter) and had high fluorescence in the FL1 channel of the FACS (excitation at 488 nm, emission at 520 nm). We have since become aware that the majority of the primary gliomasphere lines (7 of 10) used in this paper were contaminated with HEK cells expressing GFP. We had used these sphere cultures to illustrate the selection method, to measure self-renewal, to assess the expression of stemness genes and to test for tumorigenicity. As assessed by microsatellite analysis of 15 short tandem repeats, seven of the primary sphere cultures used in this paper and all of the three lines used for tumorigenicity experiments did not match their parental tissue, and their genetic profile was consistent with that of HEK cells. We further observed expression of GFP mRNA with reverse-transcription PCR in 7 of the 10 gliomasphere lines. Two of these lines (GSM-1 and OA-III-1) were traceable by microsatellite analysis to their parental tissue at low passages (2–9) but not at higher passages (>20), a result showing that contamination occurred during passage of the cells in the laboratory. We also described experiments done with cells prospectively isolated from freshly resected glioma tissues; we believe that these experiments remain valid. We discussed in the paper that these cells were rare and did not have the same high fluorescence as gliomasphere cultures. We speculated that glioma cells acquire high fluorescence under in vitro conditions. This led to our misinterpretation of the progressive increase in fluorescence of two of the gliomasphere cultures that were in fact being progressively overgrown by contaminating GFP-expressing HEK cells. All together, when we exclude HEK-contaminated cultures, we must conclude that glioma-initiating cells do not have high autofluorescence levels or acquire them during culture. Selection of a tumorigenic fraction may be possible on the basis of morphological characteristics. However, because an important part of the study was done on contaminated cells, we wish to retract the paper. We deeply apologize to the scientific community for erroneously reporting an artifactual phenomenon as well as for the delay in detecting, characterizing and reporting the error. Cross-contamination of cell cultures is likely to be a frequent problem. Routine tracing of cell lines by microsatellite analysis has been advocated before publishing work with cell lines. Our experience shows that this advice holds true also for primary cells passaged in culture. CorrIGenda and retraCtIon

Abstract 3235: Creation of novel rat models for cancer research using zinc finger nuclease (ZFN) technology

The rat resembles human physiology more closely than the mouse, partially because of its larger size. Rats are the preferred model for many disease fields, including cancer research, such as studies in breast and prostate cancers and bone metastasis. However, the lack of convenient experimental tools to manipulate the rat genome has largely limited the use of rat models, until the recent creation of the first targeted knockout rats by using zinc finger nuclease technology (Geurts et al., 2009). At SAGE Labs, we are uniquely positioned to create rat knockout models for all major disease categories. In regards to cancer research, we have developed p53 knockout rats, which may be used to significantly shorten the time required for carcinogenicity studies in the same capacity as the p53 knockout mice have been used since the mid-nineties. We are also in the process of creating a suite of immunocompromised rats, such as knockouts of RAG1, RAG2, and DNAPK, which may be used, for example, in the generation of xenografts for metastasis studies and bone marrow transplantation for cancer therapy. In addition, we have developed a set of toxicology models, including knockouts of Mdr1a, PXR, BCRP, Mrp1 and Mrp2, which can benefit studies on cancer drug delivery and metabolism. Model generation, their applications and future models will be discussed. Together, our goal is to help advance cancer research with the creation of novel rat models. Geurts, A. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3235.

Abstract B218: Lentiviral shRNA screen of human kinases identifies PLK1 as a potential therapeutic target for osteosarcoma

Induction of RNA interference in cancer cells has made possible high‐throughput genome‐wide loss‐of‐function studies using reverse genetic screening techniques. Kinases play an important role in the growth and survival of tumor cells. We aim to identify kinases that are vital to the survival of osteosarcoma cells and may be a key target in creating novel anticancer therapies. We describe an optimized systematic screen of known kinases using osteosarcoma cell lines (KHOS and U‐2OS) and a lentiviral‐based short hairpin RNA (shRNA) human kinase library. CellTiter 96® AQueous One Solution Cell Proliferation Assay was used to measure cell growth and survival. We identified several kinases, including human polo‐like kinase (PLK1), which inhibit cell growth and induce apoptosis in osteosarcoma cells when knocked down. cDNA rescue and synthetic siRNA assays confirm that the observed phenotypic changes result from the loss of PLK1 gene expression. Furthermore, a small molecule inhibitor to PLK1 inhibited osteosarcoma cell growth and induced apoptosis. Western blot analysis confirmed that PLK1 is highly expressed and activated in several osteosarcoma cell lines as well as in resected tumor samples. Immunohistochemistry analysis showed that patients with high PLK1 tumor expression levels correlated with significantly shorter survival than patients with lower levels of tumor PLK1 expression. These results demonstrate the capability and feasibility of a high‐throughput screen with a large collection of lentiviral kinases and its effectiveness in identifying potential drug targets. The development of more potent inhibitors that target PLK1 may open doors to a new range of anti‐cancer strategies in osteosarcoma. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B218.