Provided is a method for knocking in a gene of interest to a cell. The genome of the cell contains a negative selectable marker, e.g., a thymidine kinase gene flanked by a pair of recombinase recognition sites (RRS), e.g., attP. The method involves introducing into the cell a targeting construct that contains a gene of interest flanked by a second pair of RRS, e.g., attB. The targeting construct also contains in the vector backbone a negative selectable marker, e.g., thymidine kinase gene. When a recombinase recognizing the RRS is expressed, the recombination events between the two pairs of RRS result in the site-specific integration of the gene of interest in the genome of the cell. Upon selection based on the negative selectable marker, the parental cells, cells with undesired integration, e.g., random integration, or the integration of the vector backbone are removed.

The present invention provides antibodies that bind to the human glucagon receptor, designated GCGR and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human GCGR. The antibodies of the invention are useful for lowering blood glucose levels and blood ketone levels and are also useful for the treatment of diseases and disorders associated with one or more GCGR biological activities, including the treatment of diabetes, diabetic ketoacidosis and long-term complications associated with diabetes, or other metabolic disorders characterized in part by elevated blood glucose levels.

The present invention provides antibodies that bind to the human glucagon receptor, designated GCGR and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human GCGR. The antibodies of the invention are useful for lowering blood glucose levels and blood ketone levels and are also useful for the treatment of diseases and disorders associated with one or more GCGR biological activities, including the treatment of diabetes, diabetic ketoacidosis and long-term complications associated with diabetes, or other metabolic disorders characterized in part by elevated blood glucose levels.

Described herein are specific CHO genomic sites for targeted insertion of exogenous genes. The sites are located within a sequence selected from SEQ ID NOs: 1-16.

The disclosure relates to an expression method of a Haemocoagulase Acutus (Halase) recombinant protein. The method includes the following steps: (1) optimizing a halase gene; (2) performing Polymerase Chain Reaction (PCR) amplification on an optimized halase gene; (3) constructing an Agkis-pMCX expression vector, transforming plasmids to competent cells of an Escherichia coli for amplification, screening in an Amp-resistant manner for positive cloning, sequencing and extracting recombinant plasmids with correct sequencing; (4) transfecting recombinant plasmids to CHO cells; and (5) expressing the recombinant protein and identifying. According to the expression method, a recombinant halase is expressed first using the CHO cells cultured in a serum-free suspension manner; and by utilizing a bioengineering means, the actual production problems of insufficient raw material sources and unstable quality of a snake venom product are solved successfully.

The disclosure relates to an expression method of a Haemocoagulase Acutus (Halase) recombinant protein. The method includes the following steps: (1) optimizing a halase gene; (2) performing Polymerase Chain Reaction (PCR) amplification on an optimized halase gene; (3) constructing an Agkis-pMCX expression vector, transforming plasmids to competent cells of an Escherichia coli for amplification, screening in an Amp-resistant manner for positive cloning, sequencing and extracting recombinant plasmids with correct sequencing; (4) transfecting recombinant plasmids to CHO cells; and (5) expressing the recombinant protein and identifying. According to the expression method, a recombinant halase is expressed first using the CHO cells cultured in a serum-free suspension manner; and by utilizing a bioengineering means, the actual production problems of insufficient raw material sources and unstable quality of a snake venom product are solved successfully.

The present disclosure relates generally to modified adeno-associated virus (AAV) from serotypes other than serotype 2, which have a viral capsid protein with a subunit 1 (VP1) sequence which is modified relative to the corresponding wildtype sequence. In particular, the modified AAVs of the disclosure comprise site-specific amino acid substitutions within the phospholipase A2 (PLA2) domain and flanking sequence relative to the corresponding wild-type sequence which improve functionality of the AAV when produced in insect cells. The present disclosure also relates to methods of producing the modified AAVs, reagents therefor, baculovirus expression systems and insect cells for producing said modified AAVs.

Methods for introducing a scarless targeted genetic modification into a preexisting targeting vector are provided. The methods can use combinations of bacterial homologous recombination (BHR) and in vitro assembly to introduce such targeted genetic modifications into a preexisting targeting vector in a scarless manner.

Certain embodiments are directed to novel heterotrimeric fusions in which the ectodomain of the TGF-β type II receptor (TβP?II) is coupled to the N- and C-terminal ends of the endoglin-domain of the TGF-β type III receptor (TpRIIIE). Certain embodiments are directed to novel heterotrimeric polypeptides in which the ectodomain of the TGF-β type II receptor (TI3RII) is coupled to the N- and C-terminal ends of the endoglin-domain (E domain) of the TGF-β type III receptor (TI3RIII). This trimeric receptor, known as RER, can bind all three TGF-β isoforms with sub-nanomolar affinity and is effective at neutralizing signaling induced by all three TGF-β isoforms, but not other ligands of the TGF-β superfamily, such as activins, growth and differentiation factors (GDFs), and bone morphonogenetic proteins (BMPs).

Provided are methods for screening a desired variant of a target gene in a eukaryotic system. Compositions for screening a desired variant of a target gene are also provided.