FIGURE 8.1 Flowchart of gene overexpression in cells and animals.

mammalian cells. One is transient transfection in which exogenous cDNA is introduced into cells and allowed to be expressed for 1 to 3 days. The transfected cells are lysed and the proteins are analyzed. The purpose of transient transfection is usually to obtain a burst in the expression of the transferred cDNA; however, such transfection is not suitable for the selection of stably transfected cell lines. The other type is stable transfection in which foreign genes are introduced into cells and stably integrated into the chromosomes or genomes of the host cells. The integrated DNA can replicate efficiently and is maintained during cell division. The products expressed can be analyzed from successive generations of divided cells, establishing genetically altered cell lines.

Overall, gene overexpression by sense cDNA or RNA approaches consists of highly involved techniques that include isolation and characterization of a specific cDNA, DNA recombination, gene transfer and analysis of the gene expression in transfected cells or transgenic animals. The present chapter offers detailed protocols that have been successfully utilized in our laboratories.


Selection of an appropriate vector, including promoters, enhancers, selectable markers, reporter genes and poly(A) signals, is very important for the success of gene transfer and analysis of gene expression. A number of vectors are commercially available. Each has its own strengths and weaknesses with regard to gene transfection and expression. Based on our experience, we recommend and describe several plasmid-based vectors widely used in mammalian systems.



Constitutive Promoter Vectors

The anatomy of a constitutive vector is shown in Figure 8.2. The function of each component is described next.

Constitutive Promoters

The function of this type of promoter is to drive the expression of the cDNA cloned downstream from the promoter. Its driving activity is constitutive. A typical promoter is human cytomegalovirus (CMV), a type of vector that is commercially available. For example, pcDNA3 and pcDNA3.1 vectors from Invitrogen contain PCMV consisting of major intermediate early promoter and enhancer regions.

The SV40 system from Promega has also been demonstrated to be a strong expression vector. The simian virus 40 (SV40) is a small double-stranded circular DNA tumor virus with a 5.2-kb genome. This genome contains an early region encoding the tumor (T) antigen, a late region encoding the viral coat proteins, the origin of replication (ori), and enhancer elements near the ori. The ori and early region play essential roles for the expression of genes. Two divergent transcription units are produced from a single complex promoter or replication region. These viral transcripts are named "early" and "late" because of the time of maximal expression during infection. Both transcripts have introns and are polyadenylated. Using DNA manipulation technology, the SV40 genome has been mutated and fused with a plasmid such as pBR322 and generated a series of very valuable vectors used for gene transfer and expression.

Selectable Marker Genes

Stable transfection mandates a selectable marker gene in a vector so that stably transfected cells will confer resistance to drug selection. The commonly used marker genes include neor, hygr, and hyrtr. Detailed selection principles are described under the section on drug selection.

Reporter Genes

Even though a reporter gene is not required in an expression vector, it is recommended that an appropriate reporter gene be utilized in the expression constructs. This will allow use of the activity assay of the reporter gene for selection and characterization of stably transfected cells or transgenic animals. The reporter genes described next are widely utilized.

b-Galactosidase gene: Lac Z gene encoding for b-galactosidase is widely used as a reporter gene. The cell extracts of transfected cells can be directly assayed for b-galactosidase activity with spectrophotometric methods. The cells and embryos can be directly stained blue using X-gal. Tissues from adult animals can also be stained for tissue-specific expression. Therefore, we strongly recommend that this gene be constructed in transfection vectors.

CAT gene: A bacterial gene encoding for chloramphenicol acetyl transferase (CAT) has proven to be quite useful as a reporter gene for monitoring the expression of transferred genes in transfected cells or transgenic animals because eukaryotic cells contain no endogenous CAT activity. The CAT gene is isolated from the E. coli transposon, Tn9, and its coding region is fused to an appropriate promoter. CAT enzyme activity can be readily assayed by incubating the cell extracts with acetyl Co-A and 14C-chloramphenicol. This enzyme acylates the chloramphenicol, and products can be separated by thin-layer chromatography (TLC) on silica gel plates followed by autoradiography.

Luciferase gene: The luciferase gene that encodes for firefly luciferase has been isolated and widely utilized as a highly effective reporter gene. Compared with the CAT assay, the assay for luciferase activity is sensitive by more than 100-fold. It is simple, rapid and relatively inexpensive. Luciferase is a small, single polypeptide with a molecular weight of 62 kDa. Furthermore, it does not require any posttrans-lational modification for the activity. Other advantages of using the luciferase gene are that mammalian cells do not have endogenous luciferase activity, and that luciferase can produce, with very high efficiency, chemiluminescent light that can be easily detected.

b-glucuronidase (GUS) gene: Some investigators have employed uidA or the gusA gene from E. coli, which encodes for b-glucuronidase (GUS), as a reporter gene used for gene transfer. However, the disadvantage is that mammalian tissues contain endogenous GUS activity, thus making the enzyme assay more difficult. Nonetheless, the GUS reporter gene is an excellent reporter gene widely used in higher plant systems because plants do not contain detectable endogenous GUS activity.

Splicing Regions

A sequence of an appropriate polyadenylation site and a splicing region should be incorporated downstream from each individual gene in an expression vector so that individual mRNA species to be transcribed in the host cell can be spliced out. Thus, fusion proteins that are functionally different from the nonfusion or native protein of interest can be avoided.

Kozak Sequence and Enhancer Element

In order to enhance the expression of the cloned cDNA further, it is a good idea to incorporate an enhancer element upstream from the promoter. It has been reported that a Kozak sequence, GCC(A/G)CCAUGG, that includes the first codon, AUG, can increase the efficiency of translation up to 10-fold.

Inducible Promoter Vectors

Unlike constitutive promoter vectors, the expression of a cloned cDNA in an induc-ible vector requires an appropriate inducer. The general structure is shown in Figure 8.3. These vectors are commercially available. For instance, the pMAMneo (Clon-tech) contains the dexamethasone-inducible MMTV-LTR promoter linked to the RSV-LTR enhancer.

Retrovirus Vectors

It has been demonstrated that retroviruses can be used as effective vectors for gene transfer in mammalian system, especially for transient transfection.1,5 There are several advantages over other vector systems, which include (1) the retroviral genome can stably integrate into the host chromosome of the host cell and can be passed from generation to generation, thus providing an excellent vector for stable transfection; (2) retroviruses have a great range of infectivity and expression host for any animal cells via viral particles; (3) integration is site-specific with respect to the viral genome at long terminal repeats (LTRs); in other words, any DNA cloned within two LTRs will be expected to be integrated into chromosomal DNA of the host cell, which can preserve the structure of the gene intact with ease after integration; and (4) viral genomes are very plastic and manifest a high degree of natural size manipulation for DNA recombination. The drawback, however, is that the techniques are relatively sophisticated. Therefore, for successful transfection, it is



FIGURE 8.3 Diagram of an inducible expression vector.

necessary to elaborate briefly on the life cycle of retroviruses prior to describing the construction of retroviruse-based vectors for mediating gene transfer.

Retroviruses, such as Rous sarcoma virus (RSV) and Moloney murine leukemia virus (MoMLV), are RNA viruses that cause a variety of diseases (e.g., tumors) in humans. The virus has two tRNA primer molecules, two copies of genomic RNA (38S), reverse transcriptase, RNase H and integrase, which are packaged with an envelope. The viral envelope contains glycoproteins that can determine the host range of infection. As shown in Figure 8.4, when the virus or virion attaches to a cell, the viral glycoproteins in the envelope bind to specific receptors in the plasma membrane of the host cell. The bound complex facilitates the internalization of the virus that is now uncoated as it passes through the cytoplasm of the host cell. In the cytoplasm, reverse transcriptase in the viral genome catalyzes the formation of a double-stranded DNA molecule from the single-stranded virion RNA. The DNA molecule undergoes circularizing, enters the nucleus, and becomes integrated into the chromosome of the host cell, forming a provirus. Subsequently, the integrated provirus serves as the transcriptional template for mRNAs and virion genomic RNA. Interestingly, such transcription is catalyzed by the host RNA polymerase II. The mRNAs then undergo translation to produce viral proteins and enzymes using the host machinery. These components are packaged into viral core particles that move through the cytoplasm, attach to the inner side of the plasma membrane and then bud off. This cycle of infection, reverse transcription, transcription, translation, virion assembly and budding is repeated again and again, infecting new host cells.

A well-understood retrovirus is RSV. The mechanism of synthesis of double-stranded DNA intermediates (provirus) from viral RNA is unique and quite complex. The nucleotide sequence of the DNA molecule is different from that of the viral RNA. The sequence U3-R-U5, which is a combination of the 5' r-u5 segment and the cytoplasm of the host

Viral particle binds to a receptor and enters the cytoplasm of the host cell the cytoplasm of the host

Plasma membrane

\ Genomic RNA

\ Genomic RNA

\ Reverse transcription

Viral particle C^)


Viral particle C^)

FIGURE 8.4 Life cycle of a replication-competent retrovirus.

the 3' u3-r segment of the RNA, is present at 5' and 3' ends of the double-stranded DNA molecule. The U3-R-U5 is designated the long terminal repeat (LTR). This complete scheme can be divided into the eight steps shown in Figure 8.5: Step 1. One of the proline tRNA primers first anneals to the pbs region in the 5' r-u5 of the viral genome RNA. Reverse transcriptase catalyzes the extension of the tRNA primer from its 3'-OH end to the 5' end, producing a DNA fragment called 3' R'-(U5)'-tRNA. Step 2. RNase H removes the cap and poly(A) tail from the viral RNA as well as the viral r-u5 segment in the double-stranded region. Step 3. The 3' R'-(U5)'-tRNA separates from the pbs region, jumps to the 3' end of the viral RNA and forms an R'/r duplex. Step 4. The 3' R'-(U5)'-tRNA undergoes elongation up to the pbs region of the viral RNA by reverse transcriptase, producing the minus (-)strand of DNA.

Step 5. RNase H removes the u3-r from the 3' end of the viral RNA, followed by synthesis of DNA from the 3' end of the RNA via reverse transcriptase, generating the first LTR (U3-R-U5) that contains the promoter sequence. This serves as a part of the plus (+)strand of DNA. Step 6. All RNA, including the tRNA primer, is removed by RNase H. Step 7. The U3-R-U5 (PBS) separates from (U3)'R'(U5)', jumps to the 3' end of the complementary strand of DNA and forms a (PBS)/(PBS)' complex. This is an essential process for the virus because the jumping action brings the promoter in the U3 region from the 3' end to the 5' end of the plus

Cap 3 Q

Viral RNA tRNA primer S.


Extension of tRNA via reverse transcriptase tRNA primer


3' tRNA

DNA extension from 3'-end of RNA

FIGURE 8.5 Scheme for synthesis of double-stranded DNA containing two long terminal repeats (LTR) from Rouse sarcoma viral RNA.

strand of DNA. Thus, the promoter is now upstream from the coding region for gag-pol-env-src in the 38S RNA genome. Step 8. Reverse transcriptase catalyzes the extension of DNA from the 3'-termini of both strands, producing a double-stranded DNA (provirus). The LTRs at both ends of the DNA molecule contain promoter and enhance elements for transcription of the virus genome. The double-stranded DNA molecule can now become integrated into the chromosomes of the infected cell.

How can the harmful infectious retrovirus be used for gene transfer and expression? Because retroviruses can cause tumors in animals and human beings, we obviously do not want the whole virus genome to be used for gene transfer or as an expression vector. Recent DNA recombination technology makes it possible to manipulate the retrovirus genome so that it is a powerful tool for gene transfer. The simplest type of gene transfer system is one in which all or most of the gag, pol, and env genes in the provirus are deleted. Nonetheless, all of the cis-active elements such as the 5' and 3' LTRs, PBS(+), PBS(-) and psi (Y) packaging sequence are left intact. A foreign cDNA of interest or a selectable gene such as neo, gpt, dhfr, or hprt can be inserted at the initiating ATG site for gag. The expression of the inserted gene is driven by the 5' LTR. This manipulated vector is then fused with a plasmid fragment such as pBR322 containing the origin of replication (ori) and an antibiotic-resistant gene. The recombinant plasmids are propagated in a bacterial strain of E. coli. The vectors are then utilized for standard DNA-mediated transfec-tion of suitable recipient cells and stable transformants can be selected by using an appropriate antibiotic chemical such as G418. The partial viral RNA transcribed in the host cell can be further packaged into retroviral particles that become an infectious recombinant retrovirus. This can be done by cotransfecting the host cells with a helper virus that produces gag and env proteins, which can recognize the psi (Y) sequence on the recombinant transcript and become packaged to form viral particles.

The disadvantage of this strategy is that the culture supernatant contains recombinant and wild-type viruses. To overcome this problem, some vectors are constructed by deleting the Y sequence, replacing the 3'LTR with an SV40 terminator, the poly(A) signal, and fusing to the backbone of pBR322. Another type of expression vector may be constructed by deleting gag and env genes and by inserting a selectable marker gene (e.g., neo) or a reporter gene followed by polylinker sites for the insertion of the foreign gene or cDNA of interest at the ATG site for gag downstream from the 5' LTR. The 3' LTR can be replaced with the SV40 poly(A) signal downstream from the introduced cDNA or gene.

This chapter focuses on a replication-incompetent viral vector (Figure 8.6) This vector lacks gag, pol and env genes encoding virus structural proteins. These deleted viral genes are replaced by a marker gene (e.g., neo) and the cDNA of interest. cDNA to be expressed can be cloned at the multiple cloning site and be driven by the CMV promoter or other promoters such as the herpes simplex thymidine kinase promoter (TK). The major advantage of this vector is that any genes cloned between the 5'-LTR and 3'-LTR can be efficiently integrated into the chromosomes of the host cell because the LTRs contain a short sequence that facilitates the integration. The recombinant vector is then fused with the pBR322 backbone containing the

U3RU5 Promoter

U3RU5 Promoter

FIGURE 8.6 Diagram of a retrovirus expression vector.

origin of replication and the Ampr gene. The recombinant plasmids are then amplified in E. coli. The foreign gene or cDNA of interest can be cloned at the polylinker site downstream from the 5' LTR of recombinant vectors, and gene transfer can be carried out by a standard plasmid DNA-mediated procedure.


cDNA isolation and characterization are described in Chapter 3. Restriction enzyme digestion of DNA, ligation, bacterial transformation, and purification of plasmid DNA are given in Chapter 4. The subject of this section is to emphasize the following special points with respect to the preparation of sense cDNA constructs. Any mistakes will cause failure in the expression of the cloned cDNA.

1. Length of cDNA to be cloned: For the overexpression of a functional protein from a cDNA, the cDNA must include the entire coding region or ORF. In our experience, 10 to 20 base pairs in 5'UTR or 3'UTR are recommended for subcloning. Long 5'UTR or 3'UTR sequences may not be good for overexpression of the cloned cDNA.

2. Orientation of the cDNA: It is absolutely essential that the cDNA be placed in the sense orientation downstream from the promoter. In other words, the poly(T) strand or (-)strand of the cDNA should be 3' ^ 5' downstream from the driving promoter in order to make 5' ^ 3' mRNA.

3. Start or stop codon: Make sure that no start codon is between the driving promoter and the first start codon ATG of the cDNA. Otherwise, the protein to be expressed will be a fusion protein other than the protein of interest. For this reason, we strongly recommend that the plasmid construct DNA be partially sequenced at the multiple cloning site (MCS) to make sure that the cDNA is cloned at the right place without any ATG codon upstream from the first ATG codon in the cDNA. However, it is acceptable if a stop codon is present upstream from the first codon ATG because, during translation, the small subunit of ribosome will locate the ATG codon instead of the stop codon.

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