10 Генетическая инженерия (1160079), страница 7
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Within the cells, the recombinant DNA is transcribed, and the RNA is packaged. The resulting viralparticles therefore contain only the recombinant viral RNA and can actas vectors to introduce this RNA into target cells. Viral reverse transcriptase and integrase enzymes (produced by the helper virus) arealso packaged in the viral particle and are introduced into the targetcells. Once the engineered viral genome is inside a cell, these enzymescreate a DNA copy of the viral RNA genome and integrate it into a hostchromosome. The integrated recombinant DNA effectively becomes apermanent part of the chromosome because the virus lacks the genesnecessary to produce RNA copies of its genome and package them intonew virus particles. In most cases the use of recombinant retrovirusesis the best method for introducing DNA into large numbers of mammalian cells.Transformation of animal cells by any of the above techniques isproblematic for several reasons.
The foreign DNA is generally insertedat chromosomal locations that vary randomly from cell to cell. Whenthe foreign DNA contains a sequence homologous to a sequence on ahost chromosome, the introduced DNA is sometimes targeted to thatposition and integrated by homologous recombination. The nonhomologous integrants still outnumber the targeted ones, however, by factorsof 102 to 105. Some of these integration events are deleterious to the cellbecause they occur in and disrupt essential genes. Different integration sites can also greatly affect the expression of an integrated gene,because integrated genes are not transcribed equally well everywherein the genome. Another targeting problem involves the class of cell tobe transformed. If germ-line cells are altered, the alteration will bepassed on to successive generations of the organism.
If somatic cellsalone are affected, the alteration will affect only the treated animal.Despite these problems, this technology has been used extensivelyto study chromosome structure, as well as the function, regulation, andexpression of genes in eukaryotic cells. The successful introduction ofChapter 28 Recombinant DNA Technologyrecombinant DNA into an animal can again be illustrated by an experiment that altered an easily observable physical trait. The objective inthis case was to alter germ-line cells in mice to create an inheritablechange.Microinjection of DNA into the nuclei of fertilized mouse eggs canproduce efficient transformation (chromosomal integration). When theinjected eggs are introduced into a female mouse and allowed to develop, the new gene is often expressed in some of the newborn mice.Those in which the germ line has been altered can be identified bytesting their offspring. By careful breeding, a mouse line can be established in which all the mice are homozygous for the new gene or genes.Animals permanently altered in this way are called transgenic.
Thistechnology was used to introduce into mice the human growth hormone gene under the control of an inducible promoter. When fed a dietincluding the inducer, some of the mice that developed from injectedembryos grew to an unusually large size (Fig. 28-21).If mouse cells can be altered stably by recombinant DNA technology, so then can human cells. Introduction of DNA into human cellsoffers, for the first time, the potential for treating and even curinghuman genetic diseases that have been refractory to traditional therapies (Box 28-2, p. 1008).
A major technological limitation in these efforts is our overall knowledge of the cellular metabolism that underliesmany genetic diseases. As understanding improves, the ability to manipulate cellular metabolism by genetic engineering will improve. Acontribution to this understanding may be made by the internationalproject to sequence and map the entire human genome that will proceed through the 1990s. The technology needed to repair genetic defects brings with it the potential for altering human traits. Clearly, weare at a scientific crossroads that has far-reaching implications for thefuture of humankind.Figure 28-21 Cloning in mice.
The gene forhuman growth hormone was introduced into thegenome of the mouse on the right. Expression ofthe gene resulted in the greatly increased size ofthe mouse.1007Part IV Information PathwaysA Cure for Genetic DiseasesHuman gene therapy is a reality in the 1990s.The experiments are going forward with an unprecedented level of oversight and regulation bygovernments and scientific review committees.Because of the ethical issues inherent in thiswork, the objectives laid out by these review committees are narrowly defined; the experimentsmust meet strict ethical and practical criteria andare intended only to treat severe genetic disorders.First, the research is limited to somatic cells sothat a treated individual cannot pass genetic alterations to offspring.
Genetic engineering in humangerm-line cells conjures up misguided past attempts to "improve" human beings, and evokes awide range of objections on ethical grounds. Second, the risk to the patient must be outweighed bythe potential therapeutic benefit. The inherentrisk is exemplified by the possibility of random integration of DNA into a human chromosome leading to inactivation of a gene that regulates cell proliferation, effectively producing a cancer cell. Forthis reason the targets of the first gene therapytrials are among the most serious genetic diseases.Third, the target diseases must be limited to thosethat involve a known defect in a single gene, andthe normal gene must be cloned and available.Fourth, the disease must involve cells that can beisolated from a patient, altered in tissue culture,and then reimplanted in the patient.
This effectively limits the therapy to diseases involving cellsof the skin or bone marrow, although some successhas been achieved with other tissues such as liver.Fifth, the planned procedures must meet strictsafety standards in animal trials before attemptsare made with human beings.The key experimental hurdle is the efficient introduction of DNA into a sufficient number ofhuman cells in a form in which it can be expressed.Because very large numbers of cells must be transformed to have some hope of beneficial effects, research has focused on retroviral vectors (see Fig.28-20). Expression of introduced genes has beenhighly variable in animal trials. In many cases, theintroduced genes were expressed well in culture,then not at all when the cells were transferred toan animal.
New strategies for gene expression arebeing developed.Targets of human gene therapy include diseasesthat result from a functional lack of a single enzyme produced by a single gene (see Table 6-6).These include Lesch-Nyhan syndrome (p. 729),which occurs when hypoxanthine-guanine phosphoribosyltransferase is absent and results inmental retardation and severe behavioral problems. Two forms of severe immune deficiency,which result from a lack of adenosine deaminase(p.
729) or purine nucleoside phosphorylase, arealso promising candidates. Work on correctingadenosine deaminase deficiency is already welladvanced. Although these two diseases affect onlya small number of people, they are very serious(people with severe immune deficiency soon dieunless they are kept in a sterile environment), andin the case of adenosine deaminase deficiency theintroduction of the missing gene activity into bonemarrow cells does appear to have a beneficial effect.Another effort is focused on new approaches totreating cancer. Immune-system cells known to beassociated with tumors, called tumor-infiltratinglymphocytes, have been modified to produce a protein with demonstrated antitumor activity, calledtumor necrosis factor (TNF).
When reintroducedinto a cancer patient the modified cells migrate tothe tumor and the TNF they produce facilitatestumor shrinkage. Another approach is to removeand modify tumor cells themselves to produceTNF. When reintroduced into patients the modified cells stimulate the immune system to attackthe cancer cells. In animal trials this approach hasled to reduction or elimination of tumors and hasleft the animal immune to the cancer.Additional genetic disorders that involve treatment of bone marrow cells include the genetic disorders of hemoglobin—sickle-cell anemia (p. 187)and thalassemia. These represent more formidableproblems because hemoglobin is the product ofmore than one gene, and its expression must belimited to a small subfraction of bone marrow cellscalled the stem cells, which are the progenitors notonly of erythrocytes but of granulocytes, macrophages, and platelets. Potential treatment of somemore common genetic disorders must await development of methods to remove and replace cellsfrom other tissues.
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