The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

Compositions and methods for making cDNA from RNA templates, including double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA), are provided. The composition and methods include a recombinant or purified or modified or recombinant RNA dependent RNA polymerases (RdRp's). The RdRp has an amino acid sequence that shares identity to a contiguous segment of the amino acid sequence of one or more Partitiviridae virus RdRps, and includes a reverse transcriptase (RT) domain. Kits including dNTPs for use in generating cDNAs are included. The RdRps can function efficiently at lower temperatures than previously available RT enzymes. Methods of making cDNAs are provided using an RdRp are also provided. RdRps can also be used to identify candidate reverse transcriptase inhibitors.

Disclosed is a viral vector comprising (a) a cDNA of a recombinant viral RNA having a sequence of a Borna disease viral genome comprising a disrupted G gene of the Borna disease viral genome and an inserted G gene of an avian bornaviral genome, wherein the cDNA of the recombinant viral RNA has at least an N gene, an X gene, a P gene and an L gene of the Borna disease viral genome in the same order as in the Borna disease viral genome and has an inserted foreign gene; (b) DNAs encoding ribozymes; and (c) a promoter sequence, wherein (b) the DNAs encoding ribozymes are located upstream and downstream of (a) the cDNA of the recombinant viral RNA, and (a) the cDNA of the recombinant viral RNA and (b) the DNAs encoding ribozymes are located downstream of (c) the promoter sequence. The present invention can be used as a gene introduction technique that does not affect a host chromosome and can be suitable for the application in various fields, such as the treatment and prevention of brain and neurological diseases, visualization techniques of nerve cells in the field of neuroscience, etc.

This disclosure provides, among other things, a method for making a cDNA library. In some embodiments the method may comprise adding a polyA tail to the longer RNA fragments but not the shorter RNA fragments in a sample by incubating the population of RNA fragments with a polyA polymerase, wherein the reaction conditions used preferentially tail only the longer fragments but not the shorter fragments.

Simultaneous expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase; and (8) an RNA sequence that codes for the single-strand RNA binding protein.

Disclosed herein are nucleosides, nucleotides and analogs thereof, pharmaceutical compositions that include one or more of nucleosides, nucleotides and analogs thereof, and methods of synthesizing the same. Also disclosed herein are methods of ameliorating and/or treating a disease and/or a condition, including an infection from a paramyxovirus and/or an orthomyxovirus, with a nucleoside, a nucleotide and an analog thereof.

The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof.

Provided is a new-type cis-replicon construct for efficiently expressing a target protein, which comprises an RNA polymerase coding unit and a target protein coding unit. The replicon construct can drive the replication of the target protein coding unit and reduce or avoid the replication and expression of the RNA polymerase, thereby effectively improving the expression of the target protein and reducing the expression of the non-target protein.

Described are methods for increasing the expression of a transgene in eukaryotic cells by reducing RNA interference (RNAi), and variant cells produce by the method. The methods and variant cells are useful, for example, for the efficient production of therapeutic and industrial polypeptides in eukaryotic cells.