Genetically Engineering Eukaryotic Cells

Transferring genes into most eukaryotic cells is considerably more difficult than transferring them into prokaryotic cells. This is particularly true with plant and animal cells.

Vectors Used to Transfer DNA into Eukaryotic Cells

Like the vectors used for cloning DNA in bacterial cells, vectors used to transfer DNA into eukaryotic cells generally have a selectable marker and a unique restriction enzyme site. Frequently, they also have a characteristic that allows the

Vector.

lacZ'gene

Vector.

lacZ'gene

Functional lacZ' protein

Bacterial cells plated on medium containing x-gal

Figure 9.16 The Function of the lacZ' Gene in a Vector The lacZ' gene is used to differentiate cells that contain recombinant plasmid from those that contain vector alone. A functional lacZ'gene results in blue colonies when bacteria harboring intact vector are plated on a medium containing x-gal. Because disruption of the gene by an insert generates a non-functional product, cells harboring recombinant plasmids form white colonies.

Portion of lacZ'gene

Portion of lacZ'gene

Portion of lacZ'gene

mRNA

Functional lacZ' protein

Nonfunctional protein

Bacterial cells plated on medium containing x-gal

White colonies

Figure 9.16 The Function of the lacZ' Gene in a Vector The lacZ' gene is used to differentiate cells that contain recombinant plasmid from those that contain vector alone. A functional lacZ'gene results in blue colonies when bacteria harboring intact vector are plated on a medium containing x-gal. Because disruption of the gene by an insert generates a non-functional product, cells harboring recombinant plasmids form white colonies.

researcher to determine whether or not the vector has acquired an insert. Vectors are generally specific for the type of eukaryot-ic cell used as a host. For example, the vectors used to genetically engineer plants are different from those used to introduce DNA into yeast cells. Available vectors include:

■ The Ti plasmid of Agrobacterium This plasmid is unique because when the plant pathogen

A. tumefaciens comes into contact with any of a wide variety of plant cells, a portion of the Ti plasmid is transferred to that cell and is integrated into its chromosome. The transferred DNA, T-DNA, encodes proteins that cause the crown gall tumors in the plant. Scientists recognized that they could exploit the natural processes of the Ti plasmid by removing the tumor-producing genes and inserting other genes in their place. These new genes can then be transferred and integrated into the plant DNA. ■ Agrobacterium, p. 211

■ Artificial chromosomes These are segments of DNA that have the key elements of a chromosome and are stripped of other genetic information, creating sites into which large pieces of insert DNA can be cloned. Yeast artificial chromosomes can be used to clone segments of

9.6 Techniques Used in Genetic Engineering 235

DNA as large as 1,000,000 nucleotides in length into the yeast Saccharomyces cerevisiae. Most recently, mammalian artificial chromosomes have been developed that can be used to transfer DNA into animal cells. Viruses Certain viruses have been engineered for use as vectors to carry DNA into eukaryotic cells. Some of these can replicate independently of the chromosome for at least short periods of time, but they are not maintained for extended periods. Those that integrate into the DNA of the host cell remain as a permanent part of the cell. Because these vectors insert randomly into the chromosome, however, they have the undesirable characteristic of potentially disrupting the function or regulation of existing genes. ■ viruses, p. 11

Introducing DNA into Eukaryotic Cells

A vector is only useful if the DNA it carries can be delivered into a cell. An enormous advantage of using the Ti plasmid to engineer plant cells is that the T-DNA, as well as any genes inserted into it, is naturally transferred from Agrobacterium into the new host cell. Likewise, the natural ability of a virus to infect a cell can be exploited to deliver virus-derived vectors into a cell. Another method of introducing DNA into eukaryotic cells is electroporation, a method also used to introduce DNA into prokaryotic cells. The newest and perhaps most versatile method uses a gene gun, a device that uses a gas pulse or other mechanism to propel DNA-coated gold particles directly into a cell (figure 9.17).

Figure 9.17 A Gene Gun This handheld device uses a gas pulse to propel DNA-coated gold particles directly into a cell.

236 Chapter 9 Biotechnology and Recombinant DNA

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Responses

  • tommaso
    How Viruses can be used to Genetically Engineer Eukaryotic Cells?
    3 years ago
  • Makda
    What is genetic engineering in eukaryotes?
    9 days ago

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