https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/49782/1/Yamanaka_Cell_131_5.pdf

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

Embryonic Stem Cell Lines Derived from Human Blastocysts

https://www.seas.upenn.edu/%7Edischer/pdfs/Cell_on_Gel-CELLpaper.pdf

Matrix elasticity directs stem cell lineage specification.

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

/pdf/genome-engineering-using-the-crispr-cas9-system-4x8nw03mgu.pdf

Genome engineering using the CRISPR-Cas9 system

/pdf/functional-genomic-studies-of-cancer-immune-evasion-using-in-2vu31605.pdf

Functional genomic studies of cancer immune evasion using in vitro and in vivo CRISPR/Cas9 genetic screens

Modifizierte
Nukleinsaure,
umfassend eine Nukleinsaure,
kovalent gebunden an eine Verbindung mit der Formel: R 1 -X-R 2 , worin
R 1 eine Epoxidgruppe ist; worin R 2 eine Alkoxysilangruppe ist; und worin
X eine Einheit ist, welche fur
die Verknupfung
der Epoxidgruppe und der Alkoxysilangruppe chemisch geeignet ist.

Chemisch modifizierte Nukleinsäuren und Methode zur Kopplung von Nukleinsäuren an einen festen Träger

The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.

/pdf/intronization-enhances-expression-of-s-protein-and-other-3qyxv4pa6f.pdf

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

TheRoleandFateofDNA EndsforHomologous Recombination in Embryonic StemCells

The identification of cancer maintenance genes—driver genes essential to tumor survival—is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentiality in vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, and Kcnb2 knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

Allan Bradley

Papers

Functional genomic studies of cancer immune evasion using in vitro and in vivo CRISPR/Cas9 genetic screens

Chemisch modifizierte Nukleinsäuren und Methode zur Kopplung von Nukleinsäuren an einen festen Träger

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

TheRoleandFateofDNA EndsforHomologous Recombination in Embryonic StemCells

In vivo functional genomics identifies essentiality of potassium homeostasis in medulloblastoma