A C57BL/6J Fancg-KO Mouse Model Generated by CRISPR/Cas9 Partially Captures the Human Phenotype.

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.

From mice to men: GEMMs as trial patients for new NSCLC therapies.

Given the large socio-economic burden of cancer, there is an urgent need for in vivo animal cancer models that can provide a rationale for personalised therapeutic regimens that are translatable to the clinic. Recent developments in establishing mouse models that closely resemble human lung cancers involve the application of genetically engineered mouse models (GEMMs) for use in drug efficacy studies or to guide patient therapy. Here, we review recent applications of GEMMs in non-small cell lung cancer research for drug development and their potential in aiding biomarker discovery and understanding of biological mechanisms behind clinical outcomes and drug interactions.

"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

Preclinical Models of Neuroendocrine Prostate Cancer.

Prostate cancer (PCa) is the most common malignancy and the second leading cause of cancer-related death amongst men in the United States. Neuroendocrine prostate cancer (NEPC) can either arise de novo or emerge as a consequence of therapy. De novo NEPC is rare, with an incidence of <2% of all PCa cases. In contrast, treatment-induced NEPC is frequent with >20% of patients with metastatic castration-resistant prostate cancer (CRPC) reported to progress to neuroendocrine (NE) differentiation. The emergence of treatment-induced NEPC is linked to the increased therapeutic pressure, due to the broad application of androgen deprivation therapy (ADT) for PCa management and the development of novel more potent androgen receptor (AR) pathway inhibitors. NEPC is a high-grade tumor type characterized by aggressive phenotype and clinical behavior. Patients affected by NEPC frequently develop visceral metastases and have a poor prognosis. The molecular mechanisms underlying the development and progression of NEPC are still poorly understood. Transcriptional and epigenetic reprogramming appears to be involved in NE progression. In this review, we aim to provide a comprehensive view of the available models for NEPC detailing their strengths and limitations. Moreover, we describe novel approaches to expand the repertoire of preclinical models to better study, prevent, or reverse NEPC. The integration of multiple preclinical models along with molecular and omics approaches will provide important insights to understand disease progression and to devise novel therapeutic strategies for the management of NEPC in the near future. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of organoids starting from the prostate gland of a GEMM or a human PDX Basic Protocol 2: Ex vivo tumor sphere formation.

CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and the Use of Rad51 to Enhance Knock-In Allele Homozygosity via Interhomolog Repair Mechanism.

Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.

Towards Post-Meiotic Sperm Production: Genetic Insight into Human Infertility from Mouse Models.

Declined quality and quantity of sperm is currently the major cause of patients suffering from infertility. Male germ cell development is spatiotemporally regulated throughout the whole developmental process. While it has been known that exogenous factors, such as environmental exposure, diet and lifestyle, et al, play causative roles in male infertility, recent progress has revealed abundant genetic mutations tightly associated with defective male germline development. In mammals, male germ cells undergo dramatic morphological change (i.e., nuclear condensation) and chromatin remodeling during post-meiotic haploid germline development, a process termed spermiogenesis; However, the molecular machinery players and functional mechanisms have yet to be identified. To date, accumulated evidence suggests that disruption in any step of haploid germline development is likely manifested as fertility issues with low sperm count, poor sperm motility, aberrant sperm morphology or combined. With the continually declined cost of next-generation sequencing and recent progress of CRISPR/Cas9 technology, growing studies have revealed a vast number of disease-causing genetic variants associated with spermiogenic defects in both mice and humans, along with mechanistic insights partially attained and validated through genetically engineered mouse models (GEMMs). In this review, we mainly summarize genes that are functional at post-meiotic stage. Identification and characterization of deleterious genetic variants should aid in our understanding of germline development, and thereby further improve the diagnosis and treatment of male infertility.

DNA Damage Repair Deficiency in Pancreatic Ductal Adenocarcinoma: Preclinical Models and Clinical Perspectives.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and survival rates have barely improved in decades. In the era of precision medicine, treatment strategies tailored to disease mutations have revolutionized cancer therapy. Next generation sequencing has found that up to a third of all PDAC tumors contain deleterious mutations in DNA damage repair (DDR) genes, highlighting the importance of these genes in PDAC. The mechanisms by which DDR gene mutations promote tumorigenesis, therapeutic response, and subsequent resistance are still not fully understood. Therefore, an opportunity exists to elucidate these processes and to uncover relevant therapeutic drug combinations and strategies to target DDR deficiency in PDAC. However, a constraint to preclinical research is due to limitations in appropriate laboratory experimental models. Models that effectively recapitulate their original cancer tend to provide high levels of predictivity and effective translation of preclinical findings to the clinic. In this review, we outline the occurrence and role of DDR deficiency in PDAC and provide an overview of clinical trials that target these pathways and the preclinical models such as 2D cell lines, 3D organoids and mouse models [genetically engineered mouse model (GEMM), and patient-derived xenograft (PDX)] used in PDAC DDR deficiency research.

Co-Clinical Imaging Resource Program (CIRP): Bridging the Translational Divide to Advance Precision Medicine.

The National Institutes of Health's (National Cancer Institute) precision medicine initiative emphasizes the biological and molecular bases for cancer prevention and treatment. Importantly, it addresses the need for consistency in preclinical and clinical research. To overcome the translational gap in cancer treatment and prevention, the cancer research community has been transitioning toward using animal models that more fatefully recapitulate human tumor biology. There is a growing need to develop best practices in translational research, including imaging research, to better inform therapeutic choices and decision-making. Therefore, the National Cancer Institute has recently launched the Co-Clinical Imaging Research Resource Program (CIRP). Its overarching mission is to advance the practice of precision medicine by establishing consensus-based best practices for co-clinical imaging research by developing optimized state-of-the-art translational quantitative imaging methodologies to enable disease detection, risk stratification, and assessment/prediction of response to therapy. In this communication, we discuss our involvement in the CIRP, detailing key considerations including animal model selection, co-clinical study design, need for standardization of co-clinical instruments, and harmonization of preclinical and clinical quantitative imaging pipelines. An underlying emphasis in the program is to develop best practices toward reproducible, repeatable, and precise quantitative imaging biomarkers for use in translational cancer imaging and therapy. We will conclude with our thoughts on informatics needs to enable collaborative and open science research to advance precision medicine.

Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification.

Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.

Transcriptional deregulation underlying the pathogenesis of small cell lung cancer.

The discovery of recurrent alterations in genes encoding transcription regulators and chromatin modifiers is one of the most important recent developments in the study of the small cell lung cancer (SCLC) genome. With advances in models and analytical methods, the field of SCLC biology has seen remarkable progress in understanding the deregulated transcription networks linked to the tumor development and malignant progression. This review will discuss recent discoveries on the roles of RB and P53 family of tumor suppressors and MYC family of oncogenes in tumor initiation and development. It will also describe the roles of lineage-specific factors in neuroendocrine (NE) cell differentiation and homeostasis and the roles of epigenetic alterations driven by changes in NFIB and chromatin modifiers in malignant progression and chemoresistance. These recent findings have led to a model of transcriptional network in which multiple pathways converge on regulatory regions of crucial genes linked to tumor development. Validation of this model and characterization of target genes will provide critical insights into the biology of SCLC and novel strategies for tumor intervention.

Animal models of meningiomas.

Meningiomas are frequent intracranial and intraspinal tumors. They are tumors of the elderly, and meningioma growth at certain localizations, as well as recurrent tumors or primary aggressive biology may pose a therapeutic challenge. To understand the growth characteristics of meningiomas, animal models can provide insights both from a biological and therapeutical point of view. Using genetically-engineered mouse models (GEMM), it has been proven that alterations of the neurofibromatosis type 2 (NF2) gene are key steps for benign meningioma development. Aggressive meningiomas can be induced by simultaneous activation of Nf2 and the PDGF (platelet-derived growth factor)/-PDGF-Receptor (R) system, or inactivation of Tp53 and cdkn2ab in mice. However, mechanisms acting in NF2 wild-type meningiomas are poorly understood so far, because appropriate models are lacking. Xenograft models have been used either by implantation of primary cultures derived from human meningiomas, or immortalized human cell lines, respectively. While the value of primary cells is limited due to low rate of overall tumor growth and slow proliferation, xenograft approaches have been shown to be helpful for the evaluation of potential medical treatment options. Future studies must incorporate new molecular meningioma tumor drivers, as well as potential treatment options based on recurrent genetic alterations into the generation of meningioma models.

A co-clinical platform to accelerate cancer treatment optimization.

Sophistication in DNA and RNA sequencing technology is unraveling the tremendous genetic and molecular complexity of human cancer. However, the rate at which this knowledge is being translated into patient care is too slow. To this end, we have designed and implemented a new translational platform, 'The Co-Clinical Trial Project', where data obtained in genetically engineered mouse models (GEMMs) of human cancer treated with protocols identical to those of ongoing clinical trials or with therapies already established in patients serve to rapidly: (i) stratify patients in terms of response and resistance on the basis of genetic and molecular criteria; (ii) identify mechanisms responsible for tumor resistance; and (iii) evaluate the effectiveness of drug combinations to overcome such resistance based on mechanistic understanding.

The promise and challenge of ovarian cancer models.

The complexity and heterogeneity of ovarian cancer cases are difficult to reproduce in in vitro studies, which cannot adequately elucidate the molecular events involved in tumor initiation and disease metastasis. It has now become clear that, although the multiple histological subtypes of ovarian cancer are being treated with similar surgical and therapeutic approaches, they are in fact characterized by distinct phenotypes, cell of origin, and underlying key genetic and genomic alterations. Consequently, the development of more personalized treatment methodologies, which are aimed at improving patient care and prognosis, will greatly benefit from a better understanding of the key differences between various subtypes. To accomplish this, animal models of all histotypes need to be generated in order to provide accurate in vivo platforms for research and the testing of targeted treatments and immune therapies. Both genetically engineered mouse models (GEMMs) and xenograft models have the ability to further our understanding of key mechanisms facilitating tumorigenesis, and at the same time offer insight into enhanced imaging and treatment modalities. While genetic models may be better suited to examine oncogenic functions and interactions during tumorigenesis, patient-derived xenografts (PDXs) are likely a superior model to assess drug efficacy, especially in concurrent clinical trials, due to their similarity to the tumors from which they are derived. Genetic and avatar models possess great clinical utility and have both benefits and limitations. Additionally, the laying hen model, which spontaneously develops ovarian tumors, has inherent advantages for the study of epithelial ovarian cancer (EOC) and recent work champions this model especially when assessing chemoprevention strategies. While high-grade ovarian serous tumors are the most prevalent form of EOC, rarer ovarian cancer variants, such as small cell ovarian carcinoma of the hypercalcemic type and transitional cell carcinoma, or non-epithelial tumors, including germ cell tumors, will also benefit from the generation of improved models to advance our understanding of tumorigenic mechanisms and the development of selective therapeutic options.