利用Red重组系统快速构建基因打靶载体

基因敲除小鼠模型是在哺乳动物体内研究基因功能最可靠的方法之一。利用常规的分子克隆的方法构建基因打靶载体往往工作周期长,对于难度特别大的基因有时甚至无法完成打靶载体的构建。通过合理应用Red重组系统和低拷贝中间载体,利用50bp的同源重组序列直接从BAC载体中克隆了长片段的小鼠基因组序列;将得到的基因组序列再次通过重组和改造,构建了Gpr56等基因的完全敲除并带有报告基因的打靶载体,实现了打靶载体的快速构建。     

Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs)

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.     

Selection-Marker-Free Modification of the Murine β-Casein Gene Using a lox2722 Site

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the β-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.     

Beyond knockout rats: New insights into finer genome manipulation in rats

The ability to “knockout” specific genes in mice via embryonic stem (ES) cell-based gene-targeting technology has significantly enriched our understanding of gene function in normal and disease phenotypes. Improvements on this original strategy have been developed to enable the manipulation of genomes in a more sophisticated fashion with unprecedented precision. The rat is the model of choice in many areas of scientific investigation despite the lack of rat genetic toolboxes. Most recent advances of zinc finger nucleases (ZFNs) and rat ES cells are diminishing the gap between rat and mouse with respect to reverse genetic approaches. Importantly, the establishment of rat ES cell-based gene targeting technology, in combination with the unique advantages of using rats, provides new, exciting opportunities to create animal models that mimic human diseases more faithfully. We hereby report our recent results concerning finer genetic modifications in the rat, and propose their potential applications in addressing biological questions.Key words: genetic manipulation, gene targeting, conditional knockout, transgenic animal, rat model, p53 knockout rat, embryonic stem cells     

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.     

Retro-recombination screening of a mouse embryonic stem cell genomic library

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for ‘rolling out’ plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage–plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed ‘retro-recombination screening’ (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a λTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage λ into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.     

W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.     

The Natural History of Biocatalytic Mechanisms

Phylogenomic analysis of the occurrence and abundance of protein domains in proteomes has recently showed that the α/β architecture is probably the oldest fold design. This holds important implications for the origins of biochemistry. Here we explore structure-function relationships addressing the use of chemical mechanisms by ancestral enzymes. We test the hypothesis that the oldest folds used the most mechanisms. We start by tracing biocatalytic mechanisms operating in metabolic enzymes along a phylogenetic timeline of the first appearance of homologous superfamilies of protein domain structures from CATH. A total of 335 enzyme reactions were retrieved from MACiE and were mapped over fold age. We define a mechanistic step type as one of the 51 mechanistic annotations given in MACiE, and each step of each of the 335 mechanisms was described using one or more of these annotations. We find that the first two folds, the P-loop containing nucleotide triphosphate hydrolase and the NAD(P)-binding Rossmann-like homologous superfamilies, were α/β architectures responsible for introducing 35% (18/51) of the known mechanistic step types. We find that these two oldest structures in the phylogenomic analysis of protein domains introduced many mechanistic step types that were later combinatorially spread in catalytic history. The most common mechanistic step types included fundamental building blocks of enzyme chemistry: “Proton transfer,” “Bimolecular nucleophilic addition,” “Bimolecular nucleophilic substitution,” and “Unimolecular elimination by the conjugate base.” They were associated with the most ancestral fold structure typical of P-loop containing nucleotide triphosphate hydrolases. Over half of the mechanistic step types were introduced in the evolutionary timeline before the appearance of structures specific to diversified organisms, during a period of architectural diversification. The other half unfolded gradually after organismal diversification and during a period that spanned ∼2 billion years of evolutionary history.     

Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase-mediated cassette exchange

Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.     

Engineering EGFP reporter constructs into a 200 kb human β-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the β-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the β- and δ-globin genes also allowed the simultaneous generation of a δ-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the γ-globin genes and the termination codon of the β-globin gene enabled the generation of reporter constructs for both ^Aγ- and ^Gγ-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the - and β-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.     

Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy

The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted. In the method we report here, a cassette carrying in tandem the hygromycin resistance gene as a positive marker and thymidine kinase gene as a negative marker is first integrated into the locus of interest and then replaced by a null targeting fragment containing no exogenous DNA. The application of this hit-and-run strategy for removal of one allele of the CPB cysteine peptidase gene array of Leishmania infantum is described.     

Recycling selectable markers in mouse embryonic stem cells.

As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.     

Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.     

Advances on the Understanding of the Origins of Synaptic Pathology in AD

Although Alzheimer’s disease (AD) was first discovered a century ago, we are still facing a lack of definitive diagnosis during the patient’s lifetime and are unable to prescribe a curative treatment. However, the past 10 years have seen a “revamping” of the main hypothesis about AD pathogenesis and the hope to foresee possible treatment. AD is no longer considered an irreversible disease. A major refinement of the classic β-amyloid cascade describing amyloid fibrils as neurotoxins has been made to integrate the key scientific evidences demonstrating that the first pathological event occurring in AD early stages affects synaptic function and maintenance. A concept fully compatible with synapse loss being the best pathological correlate of AD rather than other described neuropathological hallmarks (amyloid plaques, neurofibrillary tangles or neuronal death). The notion that synaptic alterations might be reverted, thus offering a potential curability, was confirmed by immunotherapy experiments targeting β-amyloid protein in transgenic AD mice in which cognitive functions were improved despite no reduction in the amyloid plaques burden. The updated amyloid cascade now integrates the synapse failure triggered by soluble Aβ-oligomers. Still no consensus has been reached on the most toxic Aβ conformations, neither on their site of production nor on their extra- versus intra-cellular actions. Evidence shows that soluble Aβ oligomers or ADDLs bind selectively to neurons at their synaptic loci, and trigger major changes in synapse composition and morphology, which ultimately leads to dendritic spine loss. However, the exact mechanism is not yet fully understood but is suspected to involve some membrane receptor(s).     

An Enhanced Gene Targeting Toolkit for Drosophila: Golic+

Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer “genes” in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.     

Rapid and efficient construction of markerless deletions in the Escherichia coli genome

We have developed an improved and rapid genomic engineering procedure for the construction of custom-designed microorganisms. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via markerless deletion of selected genomic regions. The deletion process was mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (i) an arabinose-inducible promoter that drives expression of λ-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (ii) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which stimulates deletion of the introduced marker by double-strand breakage-mediated intramolecular recombination. This genomic deletion was performed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette were nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.     

Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site

Gene targeting and site-specific recombination strategies allow the precise modification of the eukaryotic genome. Many of the recombination strategies currently used, however, will introduce a selection marker gene at the modified site. DNA sequences of prokaryotic origin like vector sequences, selection marker, and reporter genes have been shown to markedly influence the regulation of the modified genomic loci. In order to avoid the insertion of excess sequences, a biphasic recombination strategy involving homologous recombination and Cre-recombinase-mediated cassette exchange (RMCE) was devised and used to insert a foreign gene into the beta-casein gene in murine embryonic stem cells. The incompatibility of the heterospecific lox sites used for the recombinase-mediated cassette exchange was found to be critical for the success of the strategy. The frequently used mutant site lox511, which differs from the natural loxP site by a single point mutation, proved unsuitable for this approach. A mutant lox site carrying two point mutations, however, was highly effective and 90% of the selected cell clones carried the desired modification. This biphasic recombination strategy allows for the efficient and precise modification of gene loci without the concomitant introduction of a selectable marker gene.     

Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.     

Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a “Painless” Lead Investigational Candidate for Therapeutic Applications

Background

Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer''s disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A “painless” human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the “painless” hNGF mutants has not been quantified.

Objectives and Results

Aiming at the therapeutic application of the “painless” hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency.

Conclusions

This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF “painless” mutants.