A consensus platform for antibody characterization.

Antibody-based research applications are critical for biological discovery. Yet there are no industry standards for comparing the performance of antibodies in various applications. We describe a knockout cell line-based antibody characterization platform, developed and approved jointly by industry and academic researchers, that enables the systematic comparison of antibody performance in western blot, immunoprecipitation and immunofluorescence.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation.

Quality control for Adeno-associated viral vector production.

Adeno-associated viral vectors (AAV) are frequently used by neuroscientists to deliver tools, such as biosensors and optogenetic and chemogenetic actuators, . Despite its widespread use, AAV vector characterization and quality control can vary between labs and viral vector cores leading to variable results and irreproducibility. This protocol describes some of the characterization and quality control assays necessary to confirm an AAV vector's titer, genomic identity, serotype and purity.

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts.

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types.

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation.

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab.

Modulation of the Fanconi anemia pathway chemically induced changes in chromatin structure.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, bone marrow failure, and atypically early-onset cancers. The FA proteins function cooperatively to repair DNA interstrand crosslinks. A major step in the activation of the pathway is the monoubiquitination of the FANCD2 and FANCI proteins, and their recruitment to chromatin-associated nuclear foci.

Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal.

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs).

Regulation of the Fanconi anemia pathway by a CUE ubiquitin-binding domain in the FANCD2 protein.

The Fanconi anemia (FA)-BRCA pathway is critical for the repair of DNA interstrand crosslinks (ICLs) and the maintenance of chromosome stability. A key step in FA-BRCA pathway activation is the covalent attachment of monoubiquitin to FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI localize in chromatin-associated nuclear foci where they interact with several well-characterized DNA repair proteins.

p21 promotes error-free replication-coupled DNA double-strand break repair.

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function.

Functional interaction between the Fanconi Anemia D2 protein and proliferating cell nuclear antigen (PCNA) via a conserved putative PCNA interaction motif.

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA.

The Fanconi anemia protein interaction network: casting a wide net.

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J.