Targeted Gene Integration Research Articles

An efficient and targeted gene integration system for high-level antibody expression

Random integration linking genomic amplification has been used to generate desired cell lines for stable and high-level expressing recombinant antibodies. But this technique is laborious, and the expression level is unpredictable due to position effects. Here, we have constructed a cell-vector system for high-level antibody expression using an FRT/FLP strategy to overcome position effects. The key is to target the FRT sequence to chromosomal locations where there is a high rate of transcription and gene amplification, and the amplified genes can be maintained. To screen desired loci with high transcriptional activity and amplifiable capacity, dual weakened markers (selectable galactosidase and amplifiable dihydrofolate reductase, DHFR) and the FRT sequence were synchronously cloned into a plasmid. After transfection of a Chinese hamster ovary host cell line with this plasmid, we selected 20 candidate cell lines from 721 individual clones. An antibody gene-targeting vector carrying an FRT-fused hygromycin gene was constructed to target antibody genes into the chromosomal FRT site by FLP recombinase. Three out of 20 cell lines can be used as host cells for site-specific recombination. By using southern blot and fluorescence in situ hybridization (FISH), a candidate engineered cell line, number 37, was chosen. It contains a single FRT-tagged locus in its genome. FISH analysis indicated that the antibody genes were all located at the original FRT-tagged locus in the genome of the gene-targeted and gene-amplified cell lines. Three kinds of recombinant antibodies were successfully expressed in candidate cell line 37. The highest producers produced more than 200 μg/ml of the antibody in 6 days of continuous culture in a spinner flask.

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7 x 10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

PBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities.

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/ loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A 'shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.

A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells.

Tight control of gene activity has been achieved in cells and transgenic organisms using the Tet regulatory systems. Unregulated basal transcription can, however, be observed whenever integration of target genes driven by promoters responsive to tetracycline controlled transcriptional activators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a non-integrated state as e.g., during transient expression, intrinsic residual transcription persists in their 'off' state, which can differ in intensity among different cell types. To efficiently repress such background activities we generated tetracycline controlled transcriptional silencers (tTS) that bind promoters responsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an activated state. Of several tTS--fusions between a modified Tet repressor and transcriptional silencing domains--tTSKid was found to be most effective in reducing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnitude. The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulation of genes carried by viral or episomal vectors.

The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase

Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of beta-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.

Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression

Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.

A system for gene cloning and manipulation in the yeast Candida glabrata

The opportunistic pathogenic yeast, Candida ( Torulopsis) glabrata, is an asexual imperfect fungus that exists largely as a haploid. Besides being a clinically important pathogen, this yeast also provides a model system for understanding basic biological mechanisms such as metal-activated metallothionein-encoding gene transcription. To facilitate molecular genetic studies in C. glabrata, we isolated a strain auxotrophic for uracil biosynthesis. The ura − mutation could be functionally complemented by the URA3 gene of Saccharomyces cerevisiae, consistent with a defect in the C. glabrata URA3 gene in this strain. We also found that the centromere-based S. cerevisiae plasmid pRS316 could stably transform and replicate in multiple copies m C. glabrata. In contrast, high-copy-number S. cerevisiae plasmids containing the 2μ. circle autonomous replication sequence were not able to replicate productively in C. glabrata. We cloned the C. glabrata URA3 gene, encoding orotidine-5'-phosphate decarboxylase, by complementation of a ura3 − strain of S. cerevisiae. The deduced amino-acid sequence is highly similar to that of the URA3 protein from S. cerevisiae. C. glabrata URA3 provides a genetic locus for targeted gene integration in C. glabrata. Integrative plasmids were constructed based on the cloned C. glabrata URA3 and are applicable for directed insertions of genes of interest at the ura3 locus through homologous recombination.