Genome Alterations and New Products of Biotechnology: -Manipulation of Animal Cell Genomes Provides Information on Chromosome Structure and Gene Expression

The transformation of animal cells by foreign genetic material offers an important mechanism for expanding our knowledge of the structure and function of animal genomes, as well as for the generation of animals with new traits. This potential has stimulated intensive research into more-sophisticated means of cloning animals. Most work of this kind requires a source of cells into which DNA can be introduced. Although intact tissues are often difficult to maintain and manipulate in vitro, many types of animal cells can be isolated and grown in the laboratory if their growth requirements are carefully met. Cells derived from a particular animal tissue and grown under appropriate tissue culture conditions can maintain their differentiated properties (for example, a hepatocyte (liver cell) remains a hepatocyte) for weeks or even months. No suitable plasmid like vector is available for introducing DNA into an animal cell, so transformation usually requires the integration of the DNA into a host-cell chromosome. The efficient delivery of DNA to a cell nucleus and integration of this DNA into a chromosome without disrupting any critical genes remain the major technical problems in the genetic engineering of animal cells.

Available methods for carrying DNA into an animal cell vary in efficiency and convenience. Some success has been achieved with spontaneous uptake of DNA or electroporation, techniques roughly comparable to the common methods used to transform bacteria. They are inefficient in animal cells, however, transforming only 1 in 100 to 10,000 cells. Microinjection—the injection of DNA directly into a nucleus, using a very fine needle—has a high success rate for skilled practitioners, but the total number of cells that can be treated is small, because each must be injected individually. The most efficient and widely used methods for transforming animal cells rely on liposomes or viral vectors. Liposomes are small vesicles consisting of a lipid bilayer that encloses an aqueous compartment (see Fig. 11–4). Liposomes that enclose a recombinant DNA molecule can be fused with the membranes of target cells to deliver DNA into the cell. The DNA sometimes reaches the nucleus, where it can integrate into a chromosome (mostly at random locations). Viral vectors are even more efficient at delivering DNA. Animal viruses have effective mechanisms for introducing their nucleic acids into cells, and several types also have mechanisms to integrate their DNA into a host-cell chromosome. Some of these, such as retroviruses (see Fig. 26–30) and adenoviruses, have been modified to serve as viral vectors to introduce foreign DNA into mammalian cells. The work on retroviral vectors illustrates some of the strategies being used (Fig. 9–32). When an engineered retrovirus enters a cell, its RNA genome is transcribed to DNA by reverse transcriptase and then integrated into the host genome by the enzyme viral integrase. Special regions of DNA are required for this procedure: long terminal repeat (LTR) sequences to integrate retroviral DNA into the host chromosome and the Ψ (psi) sequence to package the viral RNA in viral particles (see Fig. 26–30).

The gag, pol, and env genes of the retroviral genome, required for retroviral replication and assembly of viral particles, can be replaced with foreign DNA. To assemble viruses that contain the recombinant genetic information, researchers must introduce the DNA into cultured cells that are simultaneously infected with a “helper virus” that has the genes to produce viral particles but lacks the Ψ sequence required for packaging. Thus, the recombinant DNA can be transcribed and its RNA packaged into viral particles. These particles can act as vectors to introduce the recombinant RNA into target cells. Viral reverse transcriptase and integrase en zymes (produced by the helper virus) are also packaged in the viral particle and introduced into the target cells. Once the engineered viral genome is inside a cell, these enzymes create a DNA copy of the recombinant viral RNA genome and integrate it into a host chromosome. The integrated recombinant DNA then becomes a permanent part of the target cell’s chromosome and is replicated with the chromosome at every cell division. The cell itself is not endangered by the integrated viral DNA, because the recombinant virus lacks the genes needed to produce RNA copies of its genome and package them into new viral particles. The use of recombinant retro viruses is often the best method for introducing DNA into large numbers of mammalian cells.

Each type of virus has different attributes, so several classes of animal viruses are being engineered as vectors to transform mammalian cells. Adenoviruses, for example, lack a mechanism for integrating DNA into a chromosome. Recombinant DNA introduced via an adenoviral vector is therefore expressed for only a short time and then destroyed. This can be useful if the objective is transient expression of a gene. Transformation of animal cells by any of the above techniques has its problems. Introduced DNA is generally integrated into chromosomes at random locations. Even when the foreign DNA contains a sequence simi lar to a sequence in a host chromosome, allowing targeting to that position, nonhomologous integrants still outnumber the targeted ones by several orders of magnitude. If these integration events disrupt essential genes, they can sometimes alter cellular functions (most cells are diploid or polyploid, however, so an integration usually leaves at least one unaffected copy of any given gene). A particularly poor outcome would involve an integration event that inadvertently activated a gene that stimulated cell division, potentially creating a cancer cell. Although such an event was once thought to be rare, recent trials suggest it is a significant hazard (Box 9–2). Finally, the site of an integration can determine the level of expression of the integrated gene, because integrants are not transcribed equally well everywhere in the genome. Despite these challenges, the transformation of ani mal cells has been used extensively to study chromosome structure and the function, regulation, and expression of genes. The successful introduction of recombinant DNA into an animal can be illustrated by an experiment that permanently altered an easily observable inheritable physical trait. Microinjection of DNA into the nuclei of fertilized mouse eggs can produce efficient transformation (chromosomal integration). When the injected 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 by testing their offspring. By careful breeding of these mice, researchers can establish a transgenic mouse line in which all the mice are ho mozygous for the new gene or genes. This technology was used to introduce into mice the gene for human growth hormone, under the control of an inducible promoter. When the mice were fed a diet that included the inducer, some of the mice that developed from injected embryos grew to an unusually large size (Fig. 9–33). Transgenic mice have now been produced with a wide range of genetic variations, including many relevant to human dis eases and their control, pointing the way to human gene therapy (Box 9–2). A very similar approach is used to generate mice in which a particular gene has been inactivated (“knockout mice”), a way of establishing the function of the inactivated gene.

FIGURE 9–31 Glyphosate-resistant soybean plants. The photographs show two areas of a soybean field in Wisconsin. (a) Without glyphosate treatment, this part of the field is overrun with weeds. (b) Glyphosate resistant soybean plants thrive in the glyphosate-treated section of the field. Glyphosate breaks down rapidly in the environment. Agricultural use of engineered plants such as these proceeds only after considerable deliberation, balancing the extraordinary promise of the technology with the need to select new traits with care. Both science and society as a whole have a stake in ensuring that the use of the resultant plants has no adverse impact on the environment or on hu man health.

FIGURE 9–32 Use of retroviral vectors in mammalian cell cloning. A typical retroviral genome (somewhat simplified here), engineered to carry a foreign gene (pink), is added to a host-cell tissue culture. The helper virus (not shown) lacks the packaging sequence, Ψ, so its RNA transcripts cannot be packaged into viral particles, but it provides the gag, pol, and env gene products needed to package the engineered retrovirus into functional viral particles. This enables the foreign gene in the recombinant retroviral genome to be introduced efficiently into the target cells.

FIGURE 9–33 Cloning in mice. The gene for human growth hormone was introduced into the genome of the mouse on the right. Expression of the gene resulted in the unusually large size of this mouse.

اخر الاخبار

اخبار العتبة العباسية المقدسة

وفد العتبة العباسية المقدسة يزور شعبة الإعداد البدني للاطّلاع على برنامج تأهيل المنتسبين الجدد

قسم بين الحرمين يقدم جهودًا مضاعفة لإعادة المفقودات إلى الزائرين

بعد برامج تدريبية متخصصة.. متحف الكفيل يبيّن منجزات ملاكاته في فن الخاتم (الأرابيسك)

شعبة مدارس الكفيل الدينية تعقد الاجتماع التحضيري الأول لمهرجان روح النبوّة الثقافي

المزيد

الآخبار الصحية

ماذا يحدث إذا تناولنا الطعام مرة واحدة فقط في اليوم

اكتشاف عوامل خطر غير متوقعة لمرض الكبد الدهني

فيروس هانتا والسفينة الموبوءة.. تحديث من الصحة العالمية

طبيب يحسم الجدل السجائر الإلكترونية وعلاقتها بالسرطان

المزيد

الآخبار التكنلوجية

لأول مرة في الولايات المتحدة.. تشغيل مفاعل مصغر نووي يغذي الذكاء الاصطناعي بالطاقة

الكشف عن تفاصيل أول عملية ناجحة لاستعادة انتاج الحيوانات المنوية

أطلس يكشف خفايا الجسم البشري بدقة أرفع بـ50 مرة من شعر الإنسان

المتحف المصري الكبير يتحول إلى "أيقونة خضراء" بدعم ياباني

المزيد

مواضيع ذات صلة

Storage Lipids: - Triacylglycerols Are Fatty Acid Esters of Glycerol

Storage Lipids:- Fatty Acids Are Hydrocarbon Derivatives

Genome Alterations and New Products of Biotechnology: -Recombinant DNA Technology Yields New Products and Challenges

Genome Alterations and New Products of Biotechnology:- New Technologies Promise to Expedite the Discovery of New Pharmaceuticals

Genome Alterations and New Products of Biotechnology:- A Bacterial Plant Parasite Aids Cloning in Plants

From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins