A limitation of retrovirusmediated gene transfer is that. The process of insertion of their genome in to the host genome is called reverse. Background information on the engineering of retroviral vectors and retrovirus packaging lines can be found in part i in the chapter on retroviral vectors. Expressing genes in the wehi231 b cell line using retroviral mediated gene transfer 38 3. Among many gene transfer systems developed to date, the retrovirus vector mediated.
The basis of the technique is gene transfer using the retroviral vector system. One of the most important factors determining the success of these studies is the number of hscs receiving the gene of interest. In an attempt to improve the efficiency of retroviral mediated gene transfer into humanlymphocytes, weinvestigated the use ofretroviral supernatants generated in a packaging cell line derived from the gibbon ape leukemia virus galv envelope pg 9. Enhanced plasma cholesterol lowering effect of retrovirus mediated ldl receptor gene transfer to whhl rabbit liver after improved surgical technique and stimulation of hepatocyte proliferation by. Gene transfer of pdx1 may reprogram tissues other than the pancreas to make insulin and control the abnormally high blood sugar levels in diabetes. Retrovirusmediated gene transfer to purified hemopoietic. Many different organs have been considered as targets for somatic gene therapy. Kay abstract recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they.
The new dna is then incorporated into the host cell. The pg packaging cells express a hybrid retroviral genome in which the moloney. A retrovirus is a type of rna virus that inserts a copy of its genome into the dna of a host cell that it invades, thus changing the genome of that cell. Johnson cancer research unit, the walter and eliza hall institute of medical research, p.
Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropincontaining media, and transduced with amphotropic retroviral vectors carrying escherichia coli. Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing dna at the time of infection. One of the most important factors determining the success of these studies is the number of hscs receiving the gene. In three mice direct evidence of a common clone in both lymphoid and myeloid tissues was also obtained. This article throws light upon the two methods used for gene transfer in plants. Highefficiency retrovirus mediated gene transfer into the livers of mice gijsbert a. Correction of the cystic fibrosis defect in vitro by. We used escherichia coli j8galactosidase as a marker gene and demonstrate a high. Retrovirusmediated wasp gene transfer corrects defective.
Recent developments in retroviralmediated gene transduction. Retrovirus vectors constructed using the murine stem cell virus mscv backbone were used for gene transfer of the gp91 phox cdna into murine xcgd bone marrow cells. Mar 21, 2002 0002 the subject invention is directed generally to gene transfer, and more particularly to retrovirus mediated gene transfer into skin in situ. Retrovirusmediated gene transfer into human hematopoietic stem. Retrovirus vector an overview sciencedirect topics. Retrovirus are used as vectors in transfer of genetic material in a host cell. The delivery of dna into animal cells is a fundamental and established procedure. Retrovirusmediated gene transfer request pdf researchgate. Pdf direct retrovirusmediated gene transfer to the. Retrovirusmediated gene transfer to tumors springerlink. The bag vector contains both the pgalactosidase gene from escherichia 124 jeanne r.
Request pdf retrovirusmediated gene transfer in transgenic animal production, gene transfer efficiency is the limiting factor in transgenesis success rates. Us5686278a methods for enhanced retrovirusmediated gene. Among many gene transfer systems developed to date, the retrovirus vector mediated gene transfer system has been an unequalled choice in gene transfer efficiency. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator cftr cdna into cfpac1, a pancreatic adenocarcinoma cell line derived from a patient with cf that stably expresses the chloride transport abnormalities. Recombinant retrovirusmediated gene transfer to normal. Efficient retroviral mediated gene transfer into rat hepatocytes in vivo tadeusz m. This is not an example of the work produced by our essay writing service. Highefficiency retrovirusmediated gene transfer gijsbert a. How aav gene transfer works general audience duration. Gene transfer technique is used very widely both in basic research and applied biology. We have used retrovirus mediated gene transfer to demonstrate complementation of the cystic fibrosis cf defect in vitro. In rats, wehave combined retroviral mediated gene transfer with a surgical procedure in which the liver is temporarily excluded from the circulation and infected in. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo.
Somatic cell nuclear transfer is applied in the gene therapy by using gene targeting method some successes are there. Background of the invention 0003 throughout this application various publications are referenced, many in parenthesis. Lentivirusmediated gene transfer in primary t cells is. However, despite the use of retroviral gene transfer in many gene therapy trials to date, significant limitations remain. Chapter 3 using retroviral mediated gene transfer to express genes in the wehi231 b cell line 38 3. Retroviralmediated gene transfer of gp91phox into bone. We have developed a variety of retrovirus vectors and efficient packaging cell lines that have facilitated the development of efficient functional expression cloning methods. Repression of retrovirusmediated transgene expression by. Retrovirusmediated gene transfer and expression cloning. Their life cycle includes an integrated state in the dna of the host chromosome. These vectors are devoid of viral genes, are nonimmunogenic, and insert the therapeutic gene in the host chromosome ensuring its transmission to daughter cells. We investigated the efficiency of an hsvtk gene therapy for the treatment of peritoneal. The stomach cancer cells that were infected with recombinant virus expressed tnfr1 or tnfr2 and were effectively killed by recombinant tnf. We have developed a variety of retrovirus vectors and.
Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator cftr cdna into cfpac1, a pancreatic adenocarcinoma cell line derived from a patient with cf that stably expresses the chloride transport. Human stomach cancer cells that poorly expressed tnfr1 or tnfr2 were infected with this virus as an in vitro model experiment of gene therapy. Effective retrovirusmediated gene transfer in normal and. This suggests that retroviral mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain.
The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous oa1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. Retrovirusmediated gene transfer as an approach to analyze. Retrovirusmediated wasp gene transfer corrects defective actin polymerization in b cell lines from wiskottaldrich syndrome patients carrying null mutations. However, there are drawbacks to using viruses to deliver genes into cells. Retrovirus mediated gene transfer is a powerful tool that can be used to understand gene functions. Pdf centrifugal enhancement of retroviral mediated gene.
Retrovirus mediated gene transfer and expression cloning. Once inside the host cells cytoplasm, the virus uses its own reverse transcriptase enzyme to produce dna from its rna genome, the reverse of the usual pattern, thus retro backwards. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector dna sequences were analyzed either in individual spleen foci 10. Dec 10, 2012 retrovirus mediated gene transfer a retrovirus is a virus that carries its genetic material in the form of rna rather than dna. Retrovirusmediated wildtype p53 gene transfer to tumors. Direct retrovirus mediated gene transfer to the synovium of the rabbit knee. Retrovirus mediated gene transfer systems were used for transfection of 293t cells. Retroviral vectors are powerful tools for gene transfer for experimentation as well as for clinical applications. Centrifugal enhancement of retroviral mediated gene transfer.
Recombinant retrovirus mediated gene transfer to normal haemopoietic cells g. Vectors derived from retroviruses have been widely studied as tools for gene transfer into mammalian tissue in vivo. Although much progress has been made in the design of retrovirus vectors, the interactions of recombinant retrovirus with host cells remain largely elusive. Powerful tools in functional genomics toshio kitamuraa, yuko koshinoa, fumi shibataa, toshihiko okia, hideaki nakajimaa, tetsuya nosakab, and hidetoshi kumagaib divisions of acellular therapy and bhematopoietic factors, the institute of medical science, the university of tokyo, tokyo, japan. You can view samples of our professional work here. Depending upon local chromatin contexts, often influenced by cell typespecific promoter events, the retroviral. However, there are no reports, which show an ultrasound effect to retrovirus mediated gene transfer efficiency. Lymphocytes from patients with lad were exposed to cd18expressing retrovirus and enriched for cells that express cd11a and cd18 lfa1 on the cell surface. This article throws light upon the five principal methods used for creation of transgenic animals. We now show how retroviral mediated gene transfer using the lnl6 vector may resolve this issue. Retroviruses have a number of features that make them unique as gene delivery vehicles. Retrovirusmediated gene transfer of mllell transforms.
In transgenic animal production, gene transfer efficiency is the limiting factor in transgenesis success rates. The original retroviral vectors used for gene therapy were based on endogenous murine viruses. Abstract human hematopoietic stem cells genetically modified by retroviral mediated gene transfer may offer new treatment options for patients with genetic. A retroviral vector containing the wildtype p53 gene under control of a. Retrovirusmediated gene transfer is commonly used in gene therapy protocols and has the potential to provide longterm expression of the transgene. Advantages of using retroviruses as gene transfer vehicles. Abstract human hematopoietic stem cells genetically modified by retroviralmediated gene transfer may offer new treatment options for patients with genetic. Influence of the transductionselection process and of ex vivo expansion on the t cell receptor. Retrovirusmediated gene transfer into human hepatocytes mariann grossman, steven e. Gene transfer technique mybiosource learning center. Retroviral vectormediated gene transfer has been central to the development of gene therapy.
Pdf retrovirusmediated herpes simplex virus thymidine. The sirna mediated down regulation of ledgfp75 has been shown. Abstract stable gene transfer into hepatocytes might be used to compensate for a genetic deficiency affecting liver function orto deliver diffusible factors into thebloodstream. Repeated cycles of retrovirusmediated hsvtk gene transfer. Retroviral mediated transfer of the herpes simplex virus thymidine kinase hsvtk gene into malignant tumors confers drug susceptibility to the antiviral drug ganciclovir. Sep 02, 2014 gene transfer of pdx1 may reprogram tissues other than the pancreas to make insulin and control the abnormally high blood sugar levels in diabetes. Correction of cd18deficient lymphocytes by retrovirus. We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Retrovirus mediated gene transfer in primary t lymphocytes. Efficient retrovirusmediated piga gene transfer and stable. The genetic and functional abnormalities in a lymphocyte cell line from a patient with lad have been corrected by retrovirus mediated transduction of a functional cd18 gene. These results show the feasibility of retrovirus mediated gene transfer to highly purified populations of lymphomyelopoietic stem cells with longterm 6 months repopulating potential by using a supernatant infection protocol. Andre lieber, leonard meuse, brian winther, and mark a. Retroviralmediated gene transfer into hepatocytes in.
Retrovirusmediated gene transfer is a powerful tool that can be used to understand gene functions. Sep 26, 2000 the mllell fusion gene results from the translocation t11. A kasid, s morecki, p aebersold, k cornetta, k culver, s freeman, e director, m t lotze, r m blaese, w f anderson. Gene expression in mice after high efficiency retroviral. Retrovirusmediated gene transfer into canine thyroid. In gene therapy a gene that is intended for delivery is packaged into a replicationdeficient viral particle to form a viral vector. The authors have recently shown that in situ transduction of the rat 9l brain tumor following hsvtkproducer cell implantation led to tumor regression after ganciclovir. Highefficiency retrovirusmediated gene transfer gijsbert. Retroviralmediated gene transfer has also been used to transfer a marker gene so that histochemical staining can be used to identify expressing cells. The fibronectin and fibronectin fragments significantly enhance retroviral mediated gene transfer into the hematopoietic cells, particularly including committed progenitors and primitive hematopoietic stem cells. Gene transfer techniques based on the vectors used the gene transfer techniques can be divided as, nonviral methods. Howard, yoshiko murakami, masaru shibano, takashi machii, eli gilboa, yuzuru kanakura, junji takeda, taroh kinoshita, wendell f. One application for which retroviral vectors have received particular attention is gene transfer into tumor cells for treatment of cancer.
Retrovirus mediated herpes simplex virus thymidine kinase gene transfer in pancreatic cancer cell lines. Efficient retrovirusmediated piga gene transfer and. Aug 15, 2003 retrovirus mediated gene transfer in polyclonal t cells results in lower apoptosis and enhanced ex vivo cell expansion of cmvreactive cd8 t cells as compared with ebvreactive cd8 t cells. Enhanced plasma cholesterol lowering effect of retrovirus. The result of that gene transfer is called chimera. A retroviral expression vector n2 containing the selectable gene, neor, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. Experimental hematology 31 2003 10071014 retrovirus mediated gene transfer and expression cloning. Royal melbourne hospital, 3050 victoria, australia summary several of parameters seeking to increase the infection frequency of normal haemopoietic cells by. Clonogenic neuroblastoma cells in patient marrow can be transduced and the neo r gene detected by observing individual neuroblastoma cell colony growth in g418, and by polymerase chain reaction pcr of individual colonies. Recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they integrate in the host cell genome. Powerful tools in functional genomics toshio kitamuraa, yuko koshinoa, fumi shibataa, toshihiko okia, hideaki nakajimaa, tetsuya nosakab, and hidetoshi kumagaib divisions of acellular therapy and bhematopoietic factors, the institute of medical science, the university of tokyo, tokyo. Retrovirusmediated gene transfer in polyclonal t cells.
A retrovirus is type of virus that carries rna as their form genetic material. Retrovirusmediated gene transfer into canine thyroid using. Toxicity studies of retroviralmediated gene transfer for. Colocalization of retrovirus and target cells on a recombinant fibronectin fragment ch296 has been shown to significantly increase gene transfer. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator cftr cdna into cfpac1, a pancreatic adenocarcinoma cell line derived from a patient with cf that stably expresses the.
Viruses used for gene therapy to date include retrovirus, adenovirus, adenoassociated virus and herpes simplex virus. Highefficiency retroviralmediated gene transfer humanand. Highefficiency retrovirusmediated gene transfer into the livers of. The gene transfer techniques in plant genetic transformation are broadly grouped into two categories. It has become an indispensable tool for gene cloning, the study of gene function and regulation and the production of small amounts of recombinant proteins for analysis and. Of these, the moloney murine leukemia retrovirus mmlv was the first widely used gene transfer vector and was the first to be used to treat an hereditary disorder using an ex vivo strategy 80.
The goal of this study was to evaluate the impact of retroviral mediated gene transfer of gp91 phox on host defense against aspergillus fumigatus in a murine model of xcgd. Retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution. Mllell increased the proliferation of myeloid colonyforming cells in. The first production of gene targeted sheep by somatic cells nuclear transfer that encodes the recombinant human a1antitrypsin rhaat mccreath et al.
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