Methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification are disclosed. In particular, sequences of interest (e.g., genes encoding biotherapeutic proteins) are inserted proximal to selectable genes in amplifiable loci, and the transformed cells are subjected to selection to induce co-amplification of the selectable gene and the sequence of interest. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, to cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.

This invention relates to the general field of recombinant expression of polypeptides in animal cell culture. More specifically, the invention concerns improved selection of cells transfected with recombinantly engineered vectors designed to express polypeptides, in particular heteromultimeric polypeptides.

Cells and cell lines are disclosed that are able to produce therapeutic proteins, antibodies, vectors, and viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors. The cells and/or cell lines can have mutations or deletions in either one or both of the endogenous di-hydrofolate reductase (DHFR/) or glutamine synthetase (GS/) genes such that DHFR and/or GS expression or function is substantially reduced or eliminated.

A reagent for glutamine synthetase reaction comprising a chelating agent and glutamine synthetase, and a reagent for quantification of ammonia comprising a chelating agent, ATP, glutamic acid, glutamine synthetase, glucose, an oxidized NAD compound, ADP-dependent hexokinase, and glucose-6-phosphate dehydrogenase, are provided.

The present disclosure is directed to genetically engineered bacteria strains with enhanced biofuel-producing capabilities from cellulosic substrates. The bacteria strains of the present disclosure comprise an inactivated Type I glutamine synthetase gene. The present disclosure is also directed to methods of producing biofuels from cellulosic biomass using the genetically engineered bacteria strains.

Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.

The invention relates to a method for improving the yield of a plant, comprising overexpressing in said plant a glutamine synthetase, said glutamine synthetase being both constitutively overexpressed in said plant and specifically overexpressed in the bundle sheath cells of said plant. The invention also relates to means for obtaining plants with improved yield.

Methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification are disclosed. In particular, sequences of interest (e.g., genes encoding biotherapeutic proteins) are inserted proximal to selectable genes in amplifiable loci, and the transformed cells are subjected to selection to induce co-amplification of the selectable gene and the sequence of interest. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, to cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.