Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.     

Disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting

We have successfully disrupted thecftr (cystic fibrosis transmembrane conductance regulator) gene at its endogenous locus in embryonic stem cells by gene targeting. We are using a double replacement strategy to introduce subtle mutations into exon 10. We report here the first step of creating a null mutation by insertion of a functionalhprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of thecftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of thecftr targeted clones has been injected into recipient blastocysts and shown to contribute to chimaeras. The targeted clones will now be used as the starting point for a second gene targeting step to remove thehprt gene in exon 10 with the concomitant introduction of the ΔF508 mutation or other mutations.     

Efficient repetitive alteration of the mouse Huntington's disease gene by management of background in the tag and exchange gene targeting strategy

The introduction of subtle mutations to predetermined locations in the mouse genome has aided in the assessment of gene function and the precise modeling of inherited disorders. Subtle mutations can be engineered into the mouse genome by the tag and exchange gene targeting strategy (Askew et al., 1993; Stacey et al., 1994; Wu et al., 1994). This two-step method involves both a positive and a negative selection. The negative selection step typically generates a large amount of undesired background that may prevent the practical recovery of gene targeted clones (Vazquez et al., 1998). In this work we describe a strategy to effectively manage this background by calculation of a tolerable level of background for a specific targeting event, pre-screening for clones with low background, subcloning and growth of cell lines under selection. This strategy was used to repeatedly and efficiently alter the mouse Huntington's disease homologue (Hdh) resulting in an average of 15 percent of the clones having the desired modification. Analysis of the remaining background clones showed they arose de novo by a mechanism that involved physical loss of the marker rather than mutation or inactivation. We calculated the rate of loss of this marker as 8.3×10^–6 events/cell/generation. We further show that the exchanged clones retained the capacity to contribute to the mouse germline demonstrating the utility of this strategy in the production of mouse lines with Hdh variants.     

Trapping of Mammalian Promoters by Cre-lox Site-Specific Recombination

One of the challenges in human genome research is to identifythe promoter sequences which play a key role in the regulationof gene expression. We report here a new promoter trapping systemfor use with mammalian cells comprised of the following threesteps: 1) Cloning of DNA fragments into a promoter-trappingvector, 2) integration of the trapping vector into a designatedtarget in the mammalian genome using the Cre site-specific recombinase,and 3) screening of integrants for trapped promoter sequencesby activation of the luciferase gene. To assess the efficiencyof this system, lox trapping vectors containing sense tk promoter,antisense tk promoter, or a non-promoter sequence of the neogene were employed. The resulting levels of luciferase activityof the site-specific integrants were measured directly. Luciferaseactivity of the integrants can be assayed under conventionalculture conditions by simply replacing the culture medium withpotassium phosphate buffer containing luciferin. Only thoseG418^r colonies carrying the tk promoter in the normal orientationexhibited a 21- to 35-fold increase in luciferase activity overthat of the other integrants. These results indicate that thissystem is an effective means of trapping promoter sequencesfrom random mammalian genomic DNA fragments.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

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.     

Introduction of <Emphasis Type="Italic">tau</Emphasis> Mutation into Cultured Rat1-R12 Cells by Gene Targeting,Using Recombinant Adeno-Associated Virus Vector

We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1ε locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1ε locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.     

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells

Summary Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk^- cells as well as E. coli tk^- cells to the Tk⁺ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk ^- mutant of E. coli C600.     

Rescue of transfected genes from mammalian cells by functional selection in Escherichia coli

Summary We have established procedures for reisolating a transfected gene from mammalian cells by selection in Escherichia coli for the function of the gene product using the Herpes simplex virus thymidine kinase gene as a model. Rescue of the gene is accomplished by three different methods. The tk gene is recloned into a plasmid in which it is hooked up by either the lac promoter or a lac/tk hybrid promoter, or the original plasmid is cut out of the host cell DNA.As the lac/tk hybrid gene can be expressed and selected both in the mammalian and E. coli cells, this type of gene rescue allows investigations on mutagenesis and methylation processes. Additionally, it offers a simple way of studying the integration of the transfected gene into the mammalian genome.Abbreviations Ap ampicillin - FU fluorouracil - HSV Herpes simplex virus     

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.Abbreviations ES embryonic stem - neo^r neomycin resistance gene - HSV herpes simplex virus - tk thymidine kinase gene - PCR polymerase chain reaction - LIF leukemia inhibitory factor - LTP long-term potentiation - Rb retinoblastoma gene product - CF cystic fibrosis     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

Introduction of the negative selection marker into replacement vectors by a single ligation step.

Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr ^−/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr ^−/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.     

Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.     

The genetic map around the tail kinks (tk) locus on mouse Chromosome 9

Tail kinks (tk) is a classical mouse skeletal mutation, located on Chromosome (Chr) 9. As the first step for the positional cloning of the tk gene, we have established a genetic map of a region surrounding the tk locus by generating a backcross segregating for tk. From this backcross, 1004 progeny were analyzed for the coat-color phenotype of the proximally located dilute (d) gene and for the distally flanking microsatellite marker, D9Mit12. Fifty-six recombinants between d and tk and 75 recombinants between tk and D9Mit12 were identified, completing a panel of 130 recombinants including one double recombinant. This panel allowed us to map five microsatellite loci as well as d and Mod-1 with respect to tk. We show that one of the microsatellite markers mapped, D9Mit9, does not recombine at all with tk in our backcross. This indicates that the D9Mit9 locus will serve as a good starting point for a chromosomal walk to the tk gene.     

The tkNeo Gene, but Not the pgkPuro Gene, Can Influence the Ability of the β-Globin LCR to Enhance and Confer Position-Independent Expression onto the β-Globin Gene

Whether drug-selectable genes can influence expression of the β-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the β-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the β-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3′ end of the β-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3′ end of the β-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the β-globin gene linked to its LCR. The findings could partly explain why data on β-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".