Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.     

The construction of transgenic and gene knockout/knockin mouse models of human disease

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.     

A simple,rapid, efficient and inexpensive strategy for sequencing clones from cDNA libraries

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ∼ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.     

3'' Alu PCR: a simple and rapid method to isolate human polymorphic markers.

Microsatellites, such as (TG)n found at random throughout the genome, or as 3' extensions of Alu sequences are being increasingly used as genetic markers because of their pluriallelic character. The search for polymorphic microsatellites is time consuming, however, as it is necessary to sequence clones containing the microsatellites sequences in order to design specific PCR primers before testing for polymorphism, which does not always occur. We propose here a new approach to generate polymorphic markers, based on the amplification of microsatellites at the 3' end of Alu sequences, without the need for cloning or sequencing steps.     

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L^?1 M13 phage (dry-weight) was produced by the isoelectric precipitation method.     

A rapid and simple method to isolate and characterize highly polymorphic markers from the centromeric regions of the human chromosomes.

Using oligonucleotide primers complementary to the 3' ends of either the Alu or the L1Hs consensus sequences in conjunction with a primer complementary to alpha satellite subsets specific to different human chromosomes, it was possible to detect and characterize polymorphisms originating from the microsatellites which are often present downstream these repetitive elements. The methodology does not require cloning, sequencing or synthesis of specific primers. Centromeric location was confirmed by linkage analysis, in situ hybridization and sequencing. The method is proposed for the generation of polymorphic markers from all centromeric regions.     

A targeted gene knockout in Drosophila

We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.     

A novel approach to the rapid isolation and nucleotide sequencing of genomic clones.

In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique. Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1. These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing. An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.     

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.     

Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos

We describe a new strategy for producing tissue-specific cDNA libraries and subsequently identifying tissue-specific clones. This method was used to screen for cDNA clones corresponding to RNAs expressed in the Drosophila head that cannot be detected in the early embryo. RNA blots were used to assess the spatial and temporal patterns of expression of these RNAs. The ensemble of 436 head-not-embryo clones identified roughly 700 distinct RNAs that are differentially expressed in the Drosophila head. The RNA expression patterns can be classified into five major categories. it is argued that this ensemble of clones represents a large fraction of all genes differentially expressed in the adult head, but not detected in the early embryo. Many of these genes are likely to encode eye- and nervous system-specific products.     

A refined restriction map of YAC clones spanning the entire human dystrophin gene

The enormous size of the human dystrophin gene (2300 kb) has so far hindered the analysis of its organization and the characterization at the genomic level of the deletion and duplication mutations causing Duchenne or Becker muscular dystrophy. A detailed physical map of the gene locus would considerably simplify these studies. We constructed a refined, long-range restriction map of the entire human dystrophin gene, using 12 overlapping YAC clones as DNA sources. The sites for six rare cutting enzymes (SfiI, NruI, EagI, BssHII, SacII, and NotI) were mapped by partial digest analysis of YACs over a region of 2600 kb, within a level of resolution of about 10 kb. Such a map provides the first detailed representation of the physical structure of the dystrophin gene. It will be useful for mapping unlocalized exons and, eventually, for the characterization of deletions and duplications leading to disease.     

适用于分离人肠道中双歧杆菌的选择性培养基

【目的】比较并评价5种双歧杆菌选择性培养基对人源双歧杆菌的分离效果,试图筛选出一种适用于人肠道中双歧杆菌分离培养的选择性培养基。【方法】采集6份健康人粪便样品稀释涂布于5种选择性培养基上,厌氧培养后计数菌落并挑选单菌落进行鉴定。同时提取样品中细菌宏基因组DNA,应用变性梯度凝胶电泳技术(Denaturing Gel Gradient Electrophoresis,DGGE)和荧光定量PCR技术(Quantitative Polymerase Chain Reaction,q-PCR)揭示样品中双歧杆菌种类和数量,并以此为依据,客观评价上述5种选择性培养基的分离效果。【结果】BSM培养基和BLM培养基上双歧杆菌的计数结果与q-PCR的定量结果最为接近,并显著高于其它3种培养基。BLM培养基上分离到双歧杆菌的种类与DGGE图谱多样性分析的结果最为接近。【结论】BLM培养基是一种适用于人肠道中双歧杆菌分离培养的选择性培养基。     

A rapid and selective methionine oxidative modification strategy

Considering the fact that site-selective late-stage diversification of peptides and proteins remains a challenge for biochemistry, strategies targeting low-abundance natural amino acids need to be further developed. As an extremely oxidation-sensitive and low-abundance amino acid, methionine emerges as a promising target for chemo- and site-selective modification. Herein we report an efficient and highly selective modification on methionine residues by one-pot O- and N-transfer reaction, generating sulfoximine-modified peptides with near-perfect conversion within 10 min. Moreover, the great tolerance to other natural amino acids has been demonstrated in reactions with various peptide substrates. To demonstrate the generality of this protocol, we have modified natural peptides and obtained sulfoximination products with high conversion rates. This methodology provides a novel strategy as the expansion of the methionine-based peptide functionalization toolbox.     

A transient transgenic RNAi strategy for rapid characterization of gene function during embryonic development

RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for "transient transgenic" knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method.