Recombinant Cloning Technology Research Articles

Evaluation of the effectiveness of RNA silencing in the filamentous fungus Aspergillus niger using a binary vector integrated with Gateway recombination cloning technology

Evaluation of the effectiveness of RNA silencing in the filamentous fungus Aspergillus niger using a binary vector integrated with Gateway recombination cloning technology

Over expression of amphiregulin promoted malignant progression in gastric cancer

Human Amphiregulin (AREG) was found to be over expressed in many types of cancer patients including gastric cancer (GC). However, its role in GC development remained unknown. In this study, over expression of AREG in MGC803 and SGC7901 GC cells were conducted by gene transfection through utilizing gateway recombinant cloning technology. Meanwhile, AREG knocked down in BGC823 and MKN45 GC cells were achieved by lentvirus-mediated shRNA oligos infection. The results showed that over expression of AREG promoted cancer cells proliferation, invasion and migration, prevented apoptotic cell death and facilitated cell cycle progression. However, the above AREG-mediated oncogenic phenotypes were reversed while AREG was knocked down in cancer cells. Furthermore, the expression of AREG in GC cells was associated with higher level of activation of both ERK/JNK/p38 and PI3K/Akt signaling pathways, which were also diminished by AREG knocked down. Finally, the result from study in vivo showed that the silencing AREG expression by AREG knocked down inhibited tumor growth in nude mice. Collectively, the data provided a convincingly laboratory evidence that over expression of AREG promoted malignant procession via activation of ERK/JNK/p38 and PI3K/Akt signaling pathways. Thus AREG possessed an oncogenic activity and could be served as a target for gastric cancer therapy.

Construction and Characterization of Adenovirus Vectors Encoding Aspartate-β-Hydroxylase to Preliminary Application in Immunotherapy of Hepatocellular Carcinoma.

Dendritic cells (DCs) harboring tumor-associated antigen are supposed to be a potential immunotherapy for hepatocellular carcinoma (HCC). Aspartate-β-hydroxylase (AAH), an overexpressed tumor-associated cell surface protein, is considered as a promising biomarker and therapeutic target for HCC. In this study, we constructed adenovirus vector encoding AAH gene by gateway recombinant cloning technology and preliminarily explored the antitumor effects of DC vaccines harboring AAH. Firstly, the total AAH mRNA was extracted from human HCC tissues; the cDNA was amplified by RT-PCR, verified, and sequenced after TA cloning. Gateway technology was used and the obtained 18T-AAH was used as a substrate, to yield the final expression vector Ad-AAH-IRES2-EGFP. Secondly, bone marrow-derived DCs were infected by Ad-AAH-IRES2-EGFP to yield AAH-DC vaccines. Matured DCs were demonstrated by increased expression of CD11c, CD80, and MHC-II costimulatory molecules. A dramatically cell-killing effect of T lymphocytes coculturing with AAH-DCs on HepG2 HCC cell line was demonstrated by CCK-8 and FCM assays in vitro. More importantly, in an animal experiment, the lysis effect of cytotoxic T lymphocytes (CTLs) on HepG2 cells in the AAH-DC group was stronger than that in the control groups. In conclusion, the gateway recombinant cloning technology is a powerful method of constructing adenovirus vector, and the product Ad-AAH-IRES2-EGFP may present as a potential candidate for DC-based immunotherapy of HCC.

Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology.

To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia.

Conditional RNAi Using the Lentiviral GLTR System.

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X).

Site-specific recombinational cloning using gateway and in-fusion cloning schemes.

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates it from classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular commercial recombinational cloning technologies, Gateway (Life Technologies) and In-Fusion (Clontech).

Phosphorylation of Minichromosome Maintenance Protein 7 (MCM7) by Cyclin/Cyclin-dependent Kinase Affects Its Function in Cell Cycle Regulation

MCM7 is one of the subunits of the MCM2-7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. After forming the pre-replication complex (pre-RC) with other components, the MCM2-7 complex is activated by DDK/cyclin-dependent kinase to initiate DNA replication. Each subunit of the MCM2-7 complex functions differently under regulation of various kinases on the specific site, which needs to be investigated in detail. In this study, we demonstrated that MCM7 is a substrate of cyclin E/Cdk2 and can be phosphorylated on Ser-121. We found that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition, we found that MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both in vitro and in vivo. Furthermore, overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser-121 in M phase is very important for a proper mitotic exit. These data suggest that the phosphorylation of MCM7 on Ser-121 by cyclin/Cdks is involved in preventing DNA rereplication as well as in regulation of the mitotic exit.

A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology

To analyze the suitability of Gateway(®) vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway(®) destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3% total soluble protein (TSP). The presented results clearly demonstrate that the Gateway(®) recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway(®) recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.

Cancer, Viruses, and Mass Migration: Paul Berg’s Venture into Eukaryotic Biology and the Advent of Recombinant DNA Research and Technology, 1967–1980

The existing literature on the development of recombinant DNA technology and genetic engineering tends to focus on Stanley Cohen and Herbert Boyer's recombinant DNA cloning technology and its commercialization starting in the mid-1970s. Historians of science, however, have pointedly noted that experimental procedures for making recombinant DNA molecules were initially developed by Stanford biochemist Paul Berg and his colleagues, Peter Lobban and A. Dale Kaiser in the early 1970s. This paper, recognizing the uneasy disjuncture between scientific authorship and legal invention in the history of recombinant DNA technology, investigates the development of recombinant DNA technology in its full scientific context. I do so by focusing on Stanford biochemist Berg's research on the genetic regulation of higher organisms. As I hope to demonstrate, Berg's new venture reflected a mass migration of biomedical researchers as they shifted from studying prokaryotic organisms like bacteria to studying eukaryotic organisms like mammalian and human cells. It was out of this boundary crossing from prokaryotic to eukaryotic systems through virus model systems that recombinant DNA technology and other significant new research techniques and agendas emerged. Indeed, in their attempt to reconstitute 'life' as a research technology, Stanford biochemists' recombinant DNA research recast genes as a sequence that could be rewritten thorough biochemical operations. The last part of this paper shifts focus from recombinant DNA technology's academic origins to its transformation into a genetic engineering technology by examining the wide range of experimental hybridizations which occurred as techniques and knowledge circulated between Stanford biochemists and the Bay Area's experimentalists. Situating their interchange in a dense research network based at Stanford's biochemistry department, this paper helps to revise the canonized history of genetic engineering's origins that emerged during the patenting of Cohen-Boyer's recombinant DNA cloning procedures.

Recombinational Cloning

AbstractThe study of protein en masse, or functional proteomics, depends on the availability of full‐length cDNAs in appropriate expression‐ready plasmid vectors for protein expression and functional analysis. Recombinational cloning is a universal cloning technique based on site‐specific recombination that is independent of the insert DNA sequence to be cloned, which differentiates this method from the classical restriction enzyme‐based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression‐ready clones using two of the most popular recombinational cloning technologies, now commercially available from Invitrogen (Gateway) and BD Clontech (Creator).

Technologies for the study of gene and protein expression in Plasmodium.

With the imminent completion of the genome sequences of several species of Plasmodium, attention is now turning to the exploitation of these genomic sequence data for vaccine, drug and diagnostic development. Several technologies have been developed over the past decade to assist in the determination of gene and protein expression on a global scale. Of these, DNA microarrays, novel high-throughput proteomic technologies and recombinational cloning technologies are lowering the barrier to functional genomic studies in Plasmodium. Of equal importance is the capacity to manipulate, store, retrieve and analyse the tremendous quantity of data generated from these genomic studies. This paper will address the use of these technologies as well as some of the computational tools that will be ultimately required to adequately study gene and protein expression in Plasmodium.

Monitoring of antigen-specific cytolytic T lymphocytes in cancer patients receiving immunotherapy.

Recent progress in molecular and immunologic approaches to discovery of tumor-associated antigens (TAA) in humans has resulted in the characterization of a number of new epitopes (3, 23). In most cases, the success of these efforts depended on the availability of tumor-specific T-cell lines or clones, which were used as probes for isolation and biochemical characterization of TAA (10, 55). Two types of methodologies have largely been used for antigen discovery: (i) biochemical fractionation of naturally processed and presented peptides derived from major histocompatibility complex (MHC) class I molecules expressed by tumor cells (16) and (ii) expression cloning of cells transfected with cDNA libraries derived from tumor cells (54). More recent introduction of the SEREX (serological analysis of tumor antigens by recombinant cDNA expression cloning) technology (47) and of computer-based modeling of peptides that best fit the relevant MHC class I molecules expressed on tumor cells (15) further expands the list of technologies available for antigen discovery and for identification of TAA which might be therapeutically useful. SEREX is based on identification of recombinant tumor antigens by immunoglobulin G (IgG) antibodies present in the patient’s serum. To qualify for immunotherapy, e.g., as components of antitumor vaccines, TAA or their newly identified epitopes have to be immunogenic, that is, able to induce and sustain an immune response specifically targeted not only to the immunizing epitope but to the tumor itself. With the exception of the products of mutated genes, few if any TAA epitopes meet the criteria for therapeutic utility, largely because they are self-antigens rather than neo-antigens. As such, they are weakly immunogenic, and tolerance for self-epitopes in tumor-bearing hosts prevents generation of strong antitumor immune responses targeting these TAA. Most of the melanoma-derived peptides are normal differentiation antigens, which are overexpressed in tumor cells (3, 9, 23). The TAA encoded by mutated genes are the exception, of course, because they are truly new antigens, but their therapeutic usefulness is limited to individually tailored treatments that are not applicable to broad-scale immunizations. Nearly all of the known TAA epitopes are ligands for T-cell receptors (TcRs) which are clonally expressed on T lymphocytes: on CD8 1 T cells expressing TcRs for nanopeptides associated with MHC class I molecules or on CD4 1 T cells

Reconstitution of in vivo cell-mediated lympholysis responses in aged mice with interleukin 2.

Advancing age is accompanied by declining immune potential. Both humoral and cellular immune responses are diminished in aged humans and experimental animals. A major lesion preventing effective T cell-mediated responses is the lack of interleukin 2 (IL 2) synthesis in aged mice. Addition of IL 2 can effectively reconstitute in vitro T cell-dependent humoral and cell-mediated immunity. These results have been extended, demonstrating that IL 2-containing lymphokine preparations when administered with antigen can restore the ability of aged mice to generate cytotoxic T lymphocytes in vivo. Furthermore, IL 2 has been identified as the active component of these lymphokine preparations through the use of purified human IL 2 prepared by recombinant DNA and gene cloning technology. The cloned IL 2 is highly effective in enhancing the immune responses of aged animals both in vitro and in vivo.