Efficient Generation of Large‐Fragment Knock‐In Mouse Models Using 2‐Cell (2C)‐Homologous Recombination (HR)‐CRISPR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bin Gu ◽  
Eszter Posfai ◽  
Marina Gertsenstein ◽  
Janet Rossant
Keyword(s):  
Homologous Recombination ◽  
Mouse Models ◽  
Large Fragment ◽  
Efficient Generation

scholarly journals Efficient generation of mouse models with the prime editing system

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Yao Liu ◽  
Xiangyang Li ◽  
Siting He ◽  
Shuhong Huang ◽  
Chao Li ◽  
...  
Keyword(s):  
Mouse Models ◽  
Efficient Generation

Effect on response to neoadjuvant chemotherapy in high-grade serous ovarian cancer by inhibiting the GAS6/AXL pathway and inducing homologous recombination deficiency.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 6080-6080
Author(s):  
Mary M Mullen ◽  
Elena Lomonosova ◽  
Michael Driscoll Toboni ◽  
Hollie M Noia ◽  
Danny Wilke ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Homologous Recombination ◽  
Neoadjuvant Chemotherapy ◽  
Mouse Models ◽  
Poor Response ◽  
Tumor Burden ◽  
High Grade ◽  
Serous Ovarian Cancer ◽  
Brca1 And Brca2 ◽  
Response To Neoadjuvant Chemotherapy

6080 Background: Less than 10% of patients with high grade serous ovarian cancer (HGSC) have a complete pathologic response to neoadjuvant chemotherapy. We aimed to identify a biomarker predictive of response to neoadjuvant chemotherapy and to determine if GAS6/AXL inhibition with AVB500 (AVB) could increase platinum response. Methods: AVB was supplied by Aravive Biologics. HGSC tumor samples were obtained pre- and post-neoadjuvant chemotherapy. GAS6 expression was measured by tissue immunohistochemistry (IHC) and serum ELISA. Four HGSC cell lines were used for all experiments. Immunofluorescent (IF) assays targeting ɣH2AX for DNA damage, RAD51, BRCA1, and BRCA2 for homologous recombination (HR) and 53BP1 for non-homologous end joining (NHEJ) were performed. Flow cytometry was used to evaluate RPA binding. DNA fiber assays were performed. In vitro clonogenic assays were done on chemoresistant ovarian tumor cells treated with carboplatin (carbo) +/- AVB and olaparib +/- AVB. Synergy assays were analyzed using Combenefit software. Mouse models were used to evaluate the combination of carboplatin + AVB and olaparib + AVB on tumor burden. Results: Patients with high pretreatment tumor GAS6 IHC expression ( > 85%) or serum GAS6 concentrations ( > 25ng/mL) were more likely to have a poor response to neoadjuvant chemotherapy than those with low GAS6 (P = 0.002). Additionally, high GAS6 concentration was associated with decreased overall survival (24.4 months versus undefined, P = 0.009). Carbo + AVB resulted in decreased clonogenic colonies compared to carbo alone (p < 0.05). In vivo tumor mouse models treated with chemotherapy + AVB had significantly less tumor burden than those treated with chemotherapy alone (50mg vs 357mg, P = 0.003). We identified an induction in HR deficiency by a decrease in RAD51, BRCA1, and BRCA2 foci and RPA binding in cells treated with carbo + AVB compared to carbo (P < 0.05). There was increase in ɣH2AX and 53BP1 foci as well as replication fork slowing in tumor cells treated with carboplatin + AVB (P < 0.01). We also AVB and carboplatin were synergistic. Olaparib + AVB resulted in decreased clonogenic colonies (P < 0.05) and decreased tumor burden in mouse models (76mg vs 171mg, P = 0.03) compared to olaparib alone. Conclusions: GAS6 is a potential biomarker predictive of poor response to neoadjuvant chemotherapy in HGSC. Inhibition of this GAS6/AXL pathway with AVB improves sensitivity to traditional neoadjuvant chemotherapy by inducing a homologous recombination deficiency.

scholarly journals Efficient generation of functional transgenes by homologous recombination in murine zygotes

1992 ◽  
Vol 20 (6) ◽  
pp. 1259-1264 ◽  
Author(s):  
Frank R. Pieper ◽  
Ineke C.M.de Wit ◽  
Arjan C.J. Pronk ◽  
Patricia M. Kooiman ◽  
Rein Strijker ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Efficient Generation

scholarly journals Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhen Liu ◽  
Zongyang Lu ◽  
Guang Yang ◽  
Shisheng Huang ◽  
Guanglei Li ◽  
...  
Keyword(s):  
Mouse Models ◽  
Human Diseases ◽  
Base Editing ◽  
Efficient Generation

scholarly journals Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells

FEBS Letters ◽  
2016 ◽  
Vol 590 (19) ◽  
pp. 3416-3424 ◽  
Author(s):  
He Lan ◽  
Shuping Li ◽  
Zihang Guo ◽  
Hongsheng Men ◽  
Yuanyuan Wu ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Es Cells ◽  
Efficient Generation ◽  
Selection Gene

Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination

2017 ◽  
Vol 212 ◽  
pp. 238-245 ◽  
Author(s):  
James R. Butler ◽  
Rafael M.N. Santos ◽  
Gregory R. Martens ◽  
Joseph M. Ladowski ◽  
Zheng-Yu Wang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Efficient Generation

scholarly journals Mouse Models for Deciphering the Impact of Homologous Recombination on Tumorigenesis

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2083
Author(s):  
Gabriel Matos-Rodrigues ◽  
Emmanuelle Martini ◽  
Bernard S. Lopez
Keyword(s):  
Homologous Recombination ◽  
Mouse Models ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Cancer Therapies ◽  
Cancer Genes ◽  
Brca1 And Brca2 ◽  
The Impact

Homologous recombination (HR) is a fundamental evolutionarily conserved process that plays prime role(s) in genome stability maintenance through DNA repair and through the protection and resumption of arrested replication forks. Many HR genes are deregulated in cancer cells. Notably, the breast cancer genes BRCA1 and BRCA2, two important HR players, are the most frequently mutated genes in familial breast and ovarian cancer. Transgenic mice constitute powerful tools to unravel the intricate mechanisms controlling tumorigenesis in vivo. However, the genes central to HR are essential in mammals, and their knockout leads to early embryonic lethality in mice. Elaborated strategies have been developed to overcome this difficulty, enabling one to analyze the consequences of HR disruption in vivo. In this review, we first briefly present the molecular mechanisms of HR in mammalian cells to introduce each factor in the HR process. Then, we present the different mouse models of HR invalidation and the consequences of HR inactivation on tumorigenesis. Finally, we discuss the use of mouse models for the development of targeted cancer therapies as well as perspectives on the future potential for understanding the mechanisms of HR inactivation-driven tumorigenesis in vivo.

scholarly journals CtIP-mediated resection is essential for viability and can operate independently of BRCA1

2014 ◽  
Vol 211 (6) ◽  
pp. 1027-1036 ◽  
Author(s):  
Federica Polato ◽  
Elsa Callen ◽  
Nancy Wong ◽  
Robert Faryabi ◽  
Samuel Bunting ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mouse Models ◽  
Genomic Stability ◽  
Homology Search ◽  
Genome Integrity ◽  
Parp Inhibition ◽  
Dna End Resection ◽  
End Resection ◽  
Dna Ends ◽  
Deficient Mice

Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3′ ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP−/− mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.

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