Edward Weinstein

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.

Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.

Creation and Preliminary Characterization of a Leptin Knockout Rat

Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses.

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]

Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat

BackgroundEngineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.ResultsAfter microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.ConclusionThe Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation.