ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

Advanced Technologies and Automation in mES Cell Workflow.

Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. To streamline and increase the efficiency in our ES cell pipeline and decrease the timeline for mouse models produced via ES cells, automation is introduced in the pipeline. Below, we describe a novel and effective approach utilizing ddPCR, dPCR, automated DNA purification, MultiMACS, and adenovirus recombinase combined screening workflow that reduces the time between therapeutic target identification and experimental validation.

Accelerated embryonic stem cell screening with a highly efficient genotyping pipeline.

INTRODUCTION: Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. METHODS AND RESULTS: The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. Here, we optimized and functionally linked magnetic activated cell sorting (MACS) technology as well as multiplex droplet digital PCR (ddPCR) to our ES cell screening process to achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. We describe this improved workflow and illustrate the reduction in time between therapeutic target identification and experimental validation. CONCLUSION: In sum, we describe a novel and effective implementation of ddPCR, multiMACS, and adenovirus recombinase into a streamlined screening workflow that significantly reduces timelines for gene targeting in mouse ES cells.

Method for analyzing signaling networks in complex cellular systems.

Now that the human genome has been sequenced, the challenge of assigning function to human genes has become acute. Existing approaches using microarrays or proteomics frequently generate very large volumes of data not directly related to biological function, making interpretation difficult. Here, we describe a technique for integrative systems biology in which: (i) primary cells are cultured under biologically meaningful conditions; (ii) a limited number of biologically meaningful readouts are measured; and (iii) the results obtained under several different conditions are combined for analysis. Studies of human endothelial cells overexpressing different signaling molecules under multiple inflammatory conditions show that this system can capture a remarkable range of functions by a relatively small number of simple measurements. In particular, measurement of seven different protein levels by ELISA under four different conditions is capable of reconstructing pathway associations of 25 different proteins representing four known signaling pathways, implicating additional participants in the NF-kappaBorRAS/mitogen-activated protein kinase pathways and defining additional interactions between these pathways.

Sequence of Plasmodium falciparum chromosome 12.

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people every year. To stimulate basic research on the disease, and to promote the development of effective drugs and vaccines against the parasite, the complete genome of P. falciparum clone 3D7 has been sequenced, using a chromosome-by-chromosome shotgun strategy. Here we report the nucleotide sequence of the third largest of the parasite's 14 chromosomes, chromosome 12, which comprises about 10% of the 23-megabase genome. As the most (A + T)-rich (80.6%) genome sequenced to date, the P. falciparum genome presented severe problems during the assembly of primary sequence reads. We discuss the methodology that yielded a finished and fully contiguous sequence for chromosome 12. The biological implications of the sequence data are more thoroughly discussed in an accompanying Article (ref. 3).