The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

The present disclosure provides a nucleic acid sequence for regulating the transcription of a nucleic acid fragment of interest, wherein the nucleic acid sequence comprises at least 2 copies of TetO-operator sequences capable of binding to a transactivator rtTA regulatable by tetracycline or a derivative thereof, and 1 copy of minimal promoter sequence containing a TATA box sequence, and at least 1 copy of a CuO-operator sequence capable of binding to a transcription repressor CymR regulatable by cumate. The present disclosure also provides a vector and a host cell containing the nucleic acid sequence, and a method for inducing the expression of a nucleic acid fragment of interest in a host cell.

CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, since genome-wide CRISPR/Cas9 libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited due to difficulties in delivery. Here we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and novel tumor suppressor genes (TSGs), Our results demonstrate that PB can be a simple and non-viral choice for efficient in vivo delivery of CRISPR libraries.

Aspects of the invention described herein, concern approaches to make genetically modified T-cells comprising a chimeric antigen receptor for human therapy. In some alternatives, the methods utilize a selection and/or isolation of CD4+ and/or CD8+ T-cells from a mixed T-cell population, such as, peripheral blood or apheresis derived mononuclear cells. Once selected/isolated, the CD4+ and/or CD8+ T-cells are then activated, genetically modified, and propagated, preferably, in separate or isolated cultures in the presence of one or more cytokines, which support survival, engraftment and/or proliferation of the cells, as well as, preferably promoting or inducing the retention of cell surface receptors, such as CD62L, CD28, and/or CD27. Included herein are also methods of treatment, inhibition, amelioration, or elimination of a cancer by administering to a subject in need thereof, one or more types of the genetically engineered T-cells or compositions that comprise the genetically engineered T-cell prepared as described herein.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

A transgenic animal model that is suitable for the cell or tissue specific assessing of thyroid hormone (TH) action in vivo is described. The recombinant DNA construct and methods suitable to generate such an animal are also provided. The assessment of TH action is based on a reporter that is dependent on an endogenously expressed thyroid hormone receptor (TR) and coregulators of said receptor.

Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Some of the vectors are useful for tetracycline-inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and a method of producing a protein of interest, in vitro, involving the mammalian host cell.

The present invention now provides a conditional vector comprising DNA encoding for: (i) an inducible expression cassette comprising an inducible promoter operably linked to a plasmid replication region; and (ii) a selectable marker.