A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.     

蝶兰胚珠的cDNA文库构建及其特异基因表达的研究

以蝶兰(Phalaenopsis“Mt.Kaala”cv SM9108)为材料,分别提取大孢子母细胞时期胚珠和成熟胚珠的PolyA RNA,反转录成cDNA,构建起两个cDNA文库。克隆筛选采用差异杂交法。从上述两个cDNA文库中,各选择一个筛选出的cDNA,对其在植物体不同器官和不同发育时期的胚珠内的表达进行了分析。结果表明该两个cDNA均为胚珠特异,并且分别在胚珠发育的特定时期表达。推测该两个cDNA的表达受胚珠内部的不同因子调控。     

Cloning and nucleotide sequence of cDNA encoding human erythrocyte band 7 integral membrane protein

cDNA clones encoding the human erythrocyte band 7 membrane protein were isolated by immunoscreening from bone marrow and HeLa cell lambda gt 11 cDNA libraries, and their nucleotide sequences were determined. HeLa- and bone marrow cell-derived sequences were identical, except for one nucleotide; the deduced sequence of 287 amino acids was confirmed by sequence identity with peptides of the erythroid protein. Structure analysis assigned band 7 protein to the type Ib transmembrane proteins.     

兔单个植入前克隆胚胎cDNA文库的构建

人与小鼠和牛在正常胚胎植入前发育过程中基因活化的研究已经取得了长足的进展 ,但是还没有对同期克隆胚胎相关研究的报道 .利用单个家兔植入前移核重构胚胎成功地构建了MⅡ卵母细胞及发育至 4 、8 细胞期的胚胎和囊胚的特异性cDNA文库 .并用 β肌动蛋白和LAPTM4α证实这类文库是可靠的 .以 8 细胞期移核重构胚cDNA文库为例 ,随机挑取克隆进行测序分析 :其中 2 3的基因EST片段可以在GenBank或EST库中找到同源序列 ,约 1 3的EST片段属于未知的新片段 ,表明这是一类重要的新兴基因资源库 (期特异性EST库 ) .这种利用单胚胎构建cDNA文库的方法 ,解决了胚胎研究材料受限的问题 ,在时间上更加精确 ,更符合胚胎发育的规律 ,也能够更加准确地反映出一些克隆胚胎的异常表型 ,是研究早期胚胎发育基因表达以及克隆胚胎再程序化基因表达的一种有效手段 .     

Comparative analysis of genes expressed in regenerating intestine and non-eviscerated intestine of Apostichopus japonicus Selenka (Aspidochirotida: Stichopodidae) and cloning of ependymin gene

The regeneration of the intestine of sea cucumber (Apostichopus japonicus) was studied by describing historically the changes that occurred during intestine regeneration on the fifth day after chemically-induced evisceration. An expressed sequence tag (EST) analysis was undertaken to identify major genes, which might be involved in intestine regeneration of A. japonicus. Two cDNA libraries were constructed with directional cloning method, one for regenerating intestine collected on the third, fourth and fifth day after evisceration (post-evisceration, PE), and the other for the non-eviscerated (NE). A total of 730 ESTs were generated by sequencing cDNA clones from the two libraries (372 from PE and 358 from NE). The results showed that the number of genes that were involved in primary metabolism of PE library was less than that of NE library, while the number of genes involved in cell defense/immunity, cell division, cell signal transduction/communication of PE library was more than that of NE library. The results also revealed that the expression of the genes which might be involved in regeneration was enhanced to some extent after evisceration. Only about 11.54% of the sequenced clones were shared by two libraries, which provided some clues for the existence of differential gene expression between PE and NE intestines. A gene named epenAj was also characterized in this study.     

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.     

Simple and efficient subtractive hybridization screening.

A method of subtractive hybridization screening is presented that does not rely on the availability of large amounts of poly(A)+ RNA. The usefulness of this method is demonstrated by the isolation of cDNA clones that are unique or present at enhanced levels in cDNA libraries derived from a psoriatic skin cell line. A very high level of enrichment for rare cDNA sequences is possible as shown by the isolation of cDNA clones present at a frequency of less than one in one hundred thousand (0.001%). This method is particularly useful in experimental systems where the amount of RNA available is limited.     

Generation of a fluorescently labeled endogenous protein library in living human cells

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100-200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3' rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.     

人肺腺癌细胞系cDNA文库的建立

本文以Uni-ZAP XR为载体,成功地建立了人肺腺癌高、低转移细胞系的cDNA文库。文中同时也探讨了总RNA的提取和mRNA的分离,cDNA的第一条链及第二条链的合成及克隆到载体等在建立cDNA文库过程中,几个关键性的问题。该项工作的完成,为下一步在这两株细胞系cDNA之间进行消减式杂交,筛选转移相关基因奠定了坚实基础。     

Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.     

A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid.

A new procedure using an asymmetrically tailed linker-primer plasmid has been developed to prepare extremely high complexity cDNA libraries. This procedure yields plasmid primed libraries with a final form equivalent to those made by the procedure of Okayama and Berg. However, the number of steps involved in library preparation is decreased. The form of the vector is such that one end of the linearized linker-primer plasmid has a 3' terminal extension of 40 deoxythymidylate residues (the dT end). The other end has a 3' terminal extension of 10 deoxycytidylate residues (the dC end). The dC end of the plasmid is blocked to further 3' extension by a 3' phosphate group. This configuration enables one to prime first strand cDNA synthesis at the dT end, tail the 3' end of the cDNA with deoxyguanylate residues without tailing the dC end (due to the 3' phosphate block). The plasmid primed cDNA can then be self-annealed and the 3' phosphate blocking group removed during the synthesis of double stranded cDNA. The efficiency of this procedure is significantly higher than other methods (including phage based libraries): linker-primer libraries have 15 to 900-fold higher complexity than libraries prepared by other methods. A cloning efficiency of 9 x 10(8) colonies per microgram of linker-primer DNA was achieved. This method should be useful for the cloning of cDNAs corresponding to extremely rare mRNAs.     

A high-throughput alphavirus-based expression cloning system for mammalian cells

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.