hESC engineering by integrase-mediated chromosomal targeting.

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in pluripotent as well as in differentiated lineages there from.

Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases.

One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature.

Generation of platform human embryonic stem cell lines that allow efficient targeting at a predetermined genomic location.

Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency, allowing for long-term expression of transgenes. In the present work, we describe a retargeting system where we used phiC31 integrase to target a plasmid to a pseudo-attP site in the cellular genome. The integration site was mapped and the chromosomal location evaluated for potential to be transcriptionally active in differentiated cells. The target plasmid, thus inserted, carried a wild-type R4 attB site that acts as a target for further integration of expression constructs. We engineered 2 hESC lines, BG01V and H9, to contain the target and showed that genetic elements such as promoter-reporter pairs can be inserted at the target efficiently and specifically. The retargeting construct has been adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture, upon random differentiation, and directed induction into neural lineages. The system described here represents a method to rapidly assemble complex plasmid-based assay systems, controllably insert them into the hESC genome, and have them actively express in undifferentiated as well as in differentiated cells.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

AIM: Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. METHODS: The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. RESULTS: Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. CONCLUSIONS: Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Simultaneous single cell stable expression of 2-4 cDNAs in HeLaS3 using psiC31 integrase system.

An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the psiC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences. By analyzing 55 psiC31-mediated integration events with five different constructs, each carrying one, two or three tandem cDNA expression cassettes, we identified 39 pseudo attP sites in the HeLaS3 chromosomes. All these sites share a common motif containing an inverted repeat and showing a similarity to the native psiC31 attP. The 36 integration events represented 27 different pseudo attP sites, suggesting the possibility of duplicate integration of the multigene expression plasmids into different genomic loci in a single cell. We demonstrated successive introduction of two different multi-cDNA expression plasmids into definite chromosomal pseudo attP sites, attaining integration of four cDNAs of known genomic constitution at precise genomic loci of a single HeLaS3 cell. The expression levels of these several transgenes were enhanced and made equally stable and robust by inserting the cHS4 insulator between genes.

Mutational derivatives of PhiC31 integrase with increased efficiency and specificity.

phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.

Creation of engineered human embryonic stem cell lines using phiC31 integrase.

It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1alpha promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1alpha promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.

Micro RNA profiling: an easy and rapid method to screen and characterize stem cell populations.

MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells.

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, phiFC1, and phiRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of approximately 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, phiFC1, and phiRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.

Integration specificity of phage phiC31 integrase in the human genome.

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.

PhiC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa.

Patients afflicted with severe laminin 5-deficient junctional epidermolysis bullosa (JEB) often die in infancy with massive cutaneous blistering. Prior approaches to genetically correct this disorder have relied on stable integration of wild-type LAMB3 sequences, using retroviral vectors. To develop a nonviral approach to JEB gene therapy, we used the phiC31 integrase, which mediates unidirectional genomic integration of plasmids containing a specific attB site. An attB-containing laminin 5 beta3 expression plasmid was integrated into the genomes of primary keratinocytes from four unrelated, genetically characterized JEB patients. phiC31 integrase supported genomic integration into epidermal progenitor cells. Regeneration of human skin on immunedeficient mice, using these cells, produced human skin tissue with restored laminin 5 expression. Furthermore, corrected JEB tissue restored hemidesmosome formation and abolished histologic evidence of subepidermal blistering. These findings provide an approach to durable nonviral correction of JEB.

Stable nonviral genetic correction of inherited human skin disease.

Current gene-transfer technologies display limitations in achieving effective gene delivery. Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations. Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer. These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in the large COL7A1 gene. To circumvent these barriers, we used the phi C31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites'. phi C31 integrase-based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients. Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion. These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.