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Figure 81.3 Diagram of a transgene. Transgenes consist of a promoter sequence, a gene, and a polyadenylation sequence. The promoter controls the expression pattern of the gene and can either be a muscle-specific promoter or derived from a viral promoter. The gene can either be a mammalian gene of interest or a reporter gene, such as GFP or ^-galactosidase, which serves to mark transfected cells. The polyadenylation sequence provides the proper modification of the end of the produced RNA transcript needed for expression in muscle cells.

Figure 81.3 Diagram of a transgene. Transgenes consist of a promoter sequence, a gene, and a polyadenylation sequence. The promoter controls the expression pattern of the gene and can either be a muscle-specific promoter or derived from a viral promoter. The gene can either be a mammalian gene of interest or a reporter gene, such as GFP or ^-galactosidase, which serves to mark transfected cells. The polyadenylation sequence provides the proper modification of the end of the produced RNA transcript needed for expression in muscle cells.

promoter, which offer high expression levels but are not muscle specific. Marker genes can be obtained from commercial sources or freely available plasmids. Genes of interest can be obtained as cDNA from commercial clone libraries or be produced from cells by techniques such as polymerase chain reaction (PCR).

Transfection can be performed either in cell culture or in live animals. For cell culture, transfection can be performed by isolating satellite cells from mice and transfecting them in vitro using a retroviral vector (Rando and Blau, 1994). Transfected cells are identified and expanded in cell culture before being injected into recipient mice (Rando and Blau, 1994). These injected cells can differentiate and join existing muscles. This approach is well suited for studying the biology and function of satellite cells in mice.

For live animals, intact muscles can be transfected in vivo either with the use of electroporation or viral infection. Electroporation uses electric pulses to permea-bilize muscle fibers and thus facilitate the entry of foreign DNA (Mir et al., 1999). The DNA is maintained as nuclear extrachromosomal DNA, which is transcription-ally active for long periods of time. This approach uses transgenes easily produced by standard molecular biology techniques but does require the use of electroporation equipment to deliver the electric pulses. Viral infection uses specific viral vectors that can be packaged into viral particles in specialized packaging cell lines (Gregorevic et al., 2004). Depending upon the viral vector, either transient or stable infection is possible. Transient infection produces transgene expression for a short period of time, which is dependent on the rate of viral clearance. Stable infection is durable due to integration of the transgene. The efficiency of infection is usually superior for viral vectors producing transient expression, but transient expression demonstrates decreasing efficiency with repeated use due to immunization of the animal.

Transgenic animals The genomic DNA of both rats and mice can be modified through the construction of transgenic animals. Trans-genic technology allows genes to be deleted (also referred to as a "knock-out") or modified (also referred to as a "knock-in") (see Figure 81.4).

The modified genes can have either changes to the amino acid sequence of the protein or changes to the

Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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