A multiauxotrophic CHO cell line for the rapid isolation of producers of diverse or high levels of recombinant proteins.

Chinese Hamster Ovary (CHO) cells are widely used for the high-level production of recombinant proteins. We created a multiauxotrophic mutant of CHO-K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA-based genes for the eight missing activities.

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively).

Saturation mutagenesis reveals manifold determinants of exon definition.

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells.

Correction of point mutations at the endogenous locus of the dihydrofolate reductase gene using repair-PolyPurine Reverse Hoogsteen hairpins in mammalian cells.

Correction of point mutations that lead to aberrant transcripts, often with pathological consequences, has been the focus of considerable research. In this work, repair-PPRHs are shown to be a new powerful tool for gene correction. A repair-PPRH consists of a PolyPurine Reverse Hoogsteen hairpin core bearing an extension sequence at one end, homologous to the DNA strand to be repaired but containing the wild type nucleotide instead of the mutation.

CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining.

Mammalian cells are widely used for the production of therapeutic recombinant proteins, as these cells facilitate accurate folding and post-translational modifications often essential for optimum activity. Targeted insertion of a plasmid harboring a gene of interest into the genome of mammalian cells for the expression of a desired protein is a key step in production of such biologics. Here we show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR-Cas9 system can be efficiently used to target ∼5 kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ).

Splicing of designer exons informs a biophysical model for exon definition.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither.

The isolation of CHO cells with a site conferring a high and reproducible transgene amplification rate.

Co-amplification of transgenes using the dihydrofolate reductase/methotrexate (DHFR/MTX) system is a widely used method for the isolation of Chinese hamster ovary (CHO) cell lines that secrete high levels of recombinant proteins. A bottleneck in this process is the stepwise selection for MTX resistant populations; which can be slow, tedious and erratic. We sought to speed up and regularize this process by isolating dhfr(-) CHO cell lines capable of integrating a transgene of interest into a defined chromosomal location that supports a high rate of gene amplification.

Quantitative evaluation of all hexamers as exonic splicing elements.

We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule.

Context-dependent splicing regulation: exon definition, co-occurring motif pairs and tissue specificity.

Splicing is a crucial process in gene expression in higher organisms because: 1) most vertebrate genes contain introns; and 2) alternative splicing is primarily responsible for increasing proteomic complexity and functional diversity. Intron definition, the coordination across an intron, is a mandatory step in the splicing process. However, exon definition, the coordination across an exon, is also thought to be required for the splicing of most vertebrate exons.

Intronic motif pairs cooperate across exons to promote pre-mRNA splicing.

Background: A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions.

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system.

Demand is increasing for therapeutic biopharmaceuticals such as monoclonal antibodies. Achieving maximum production of these recombinant proteins under developmental time constraints has been a recent focus of study. The majority of these drugs are currently produced in altered Chinese hamster ovary (CHO) cells due to the high viability and the high densities achieved by these cells in suspension cultures.

Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing.

Pre-messengerRNA (mRNA) splicing requires the accurate recognition of splice sites by the cellular RNA processing machinery. In addition to sequences that comprise the branchpoint and the 3' and 5' splice sites, the cellular splicing machinery relies on additional information in the form of exonic and intronic splicing enhancer and silencer sequences. The high abundance of these motifs makes it difficult to investigate their effects using standard genetic perturbations, since their disruption often leads to the formation of yet new elements.

Dynamic regulation of alternative splicing by silencers that modulate 5' splice site competition.

Alternative splicing makes a major contribution to proteomic diversity in higher eukaryotes with approximately 70% of genes encoding two or more isoforms. In most cases, the molecular mechanisms responsible for splice site choice remain poorly understood. Here, we used a randomization-selection approach in vitro to identify sequence elements that could silence a proximal strong 5' splice site located downstream of a weakened 5' splice site.

Searching for splicing motifs.

Intron removal during pre-mRNA splicing in higher eukaryotes requires the accurate identification of the two splice sites at the ends of the exons, or exon definition. The sequences constituting the splice sites provide insufficient information to distinguish true splice sites from the greater number of false splice sites that populate transcripts. Additional information used for exon recognition resides in a large number of positively or negatively acting elements that lie both within exons and in the adjacent introns.

Positive selection acting on splicing motifs reflects compensatory evolution.

We have used comparative genomics to characterize the evolutionary behavior of predicted splicing regulatory motifs. Using base substitution rates in intronic regions as a calibrator for neutral change, we found a strong avoidance of synonymous substitutions that disrupt predicted exonic splicing enhancers or create predicted exonic splicing silencers. These results attest to the functionality of the hexameric motif set used and suggest that they are subject to purifying selection.

Comparison of multiple vertebrate genomes reveals the birth and evolution of human exons.

Orthologous gene structures in eight vertebrate species were compared on a genomic scale to detect the birth and maturation of new internal exons during the course of evolution. We found that 40% of new human exons are alternatively spliced, and most of these are cassette exons (exons that are either included or skipped in their entirety) with low inclusion rates. This proportion decreases steadily as older and older exons are examined, even as splicing efficiency increases.

Computational searches for splicing signals.

The removal of introns from pre-mRNA requires as an initial event the accurate molecular recognition of the proper exon-intron borders. It is now evident that RNA sequence elements in addition to the consensus splice site sequences themselves are required for this recognition. Genomic analyses have contributed to the definition of these elements as exonic and intronic splicing enhancers and silencers, comprising what has been called the "splicing code.

Exon inclusion is dependent on predictable exonic splicing enhancers.

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L.

Dichotomous splicing signals in exon flanks.

Intronic elements flanking the splice-site consensus sequences are thought to play a role in pre-mRNA splicing. However, the generality of this role, the catalog of effective sequences, and the mechanisms involved are still lacking. Using molecular genetic tests, we first showed that the approximately 50-nt intronic flanking sequences of exons beyond the splice-site consensus are generally important for splicing.

Computational definition of sequence motifs governing constitutive exon splicing.

We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers.

Latent splice sites and stop codons revisited.

The accuracy of the data we reported in an RNA Letter to the Editor earlier this year on the possible relationship between stop codons and splicing is questioned by Miriami et al. (this issue). We reply here that we see no inaccuracy in our data presentation and offer a possible explanation for their interpretation.

Sequence information for the splicing of human pre-mRNA identified by support vector machine classification.

Vertebrate pre-mRNA transcripts contain many sequences that resemble splice sites on the basis of agreement to the consensus,yet these more numerous false splice sites are usually completely ignored by the cellular splicing machinery. Even at the level of exon definition,pseudo exons defined by such false splices sites outnumber real exons by an order of magnitude. We used a support vector machine to discover sequence information that could be used to distinguish real exons from pseudo exons.

Effects of genomic context and chromatin structure on transcription-coupled and global genomic repair in mammalian cells.

It has been long recognized that in mammalian cells, DNA damage is preferentially repaired in the transcribed strand of transcriptionally active genes. However, recently, we found that in Chinese hamster ovary (CHO) cells, UV-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in both the transcribed and the non-transcribed strand of exon 1 of the dihydrofolate reductase (DHFR) gene. We mapped CPD repair at the nucleotide level in the transcriptionally active DHFR gene and the adjacent upstream OST gene, both of which have been translocated to two chromosomal positions that differ from their normal endogeneous positions.

The effect of nonsense codons on splicing: a genomic analysis.

The phenomenon of nonsense-associated altered splicing raises the possibility that the recognition of in-frame nonsense codons is used generally for exon identification during pre-mRNA splicing. However, nonsense codon frequencies in pseudo exons and in regions flanking 5' splice sites are no greater than that expected by chance, arguing against the widespread use of this strategy as a means of rejecting potential splice sites.