Genes VII

The procedure for introducing exogenous donor DNA into recipient cells is called transfection. Transfection experiments began with the addition of preparations of metaphase chromosomes to cell suspensions. The chromosomes are taken up rather inefficiently by the cells and give rise to unstable variants at a low frequency. Intact chromosomes rarely survive the procedure; the recipient cell usually gains a fragment of a donor chromosome (which is unstable because it lacks a centromere). Rare cases of stable lines may have resulted from integration of donor material into a resident chromosome.

Similar results are obtained when purified DNA is added to a recipient cell preparation. However, with purified DNA it is possible to add particular sequences instead of relying on random fragmentation of chromosomes. Transfection with DNA yields stable as well as unstable lines, with the former relatively predominant. (These experiments are directly analogous to those performed in bacterial transformation, but are described as transfection because of the historiCal use of transformation to describe changes that allow unrestrained growth of eukaryotic cells.)

Unstable transfectants (sometimes called transient transfectants) reflect the survival of the transfected DNA in extrachromosomal form; stable lines result from integration into the genome. The transfected DNA can be expressed in both cases. However, the low frequencies of transfection make it necessary to use donor markers whose presence in the recipient cells can be selected for. Most transfection experiments have used markers representing readily assayed enzymatic functions, but, in principle, any marker that can be selected can be assayed. This allows the isolation of genes responsible for morphological phenomena. Most notably, transfected cells can be selected for acquisition of the transformed (tumorigenic) phenotype. This type of protocol has led to the isolation of several cellular onc genes (discussed in 28 Oncogenes and cancer).

Cotransfection with more than one marker has proved informative about the events involved in transfection and extends the range of questions that we can ask with this technique. A common marker used in such experiments is the tk gene, coding for the enzyme thymidine kinase, which catalyzes an essential step in the provision of thymidine triphosphate as a precursor for DNA synthesis.

When tk ^V cells are transfected with a DNA preparation containing both a purified tk⁺gene and the φX174 genome, all the tk⁺ transformants have both donor sequences. This is a useful observation, because it allows unselected markers to be introduced routinely by cotransfection with a selected marker.

The arrangement of tk and φX174 sequences is different in each transfected line, but remains the same during propagation of that line. Often multiple copies of the donor sequences are present, the number varying with the individual line. Revertants lose the φX174 sequences together with tk sequences. Amplification of transfected sequences under selective pressure results in the increase of copy number of all donor sequences pari passu. So the two types of donor sequence become physically linked during transfection and suffer the same fate thereafter.

To perform a transfection experiment, the mass of DNA added to the recipient cells is increased by including an excess of "carrier DNA," a preparation of some other DNA (often from salmon sperm). Transfected cells prove to have sequences of the carrier DNA flanking the selected sequences on either side. Transfection therefore appears to be mediated by a large unit, consisting of a linked array of all sequences present in the donor preparation.

Since revertants for the selected marker lose all of this material, it seems likely that the transfected cell gains only a single large unit. The unit is formed by a concatemeric linkage of donor sequences in a reaction that is rapid relative to the other events involved in transfection. This transfecting package is ~1000 kb in length.

Because of the size of the donor unit, we cannot tell from blotting experiments whether it is physically linked to recipient chromosomal DNA (the relevant end fragments are present in too small a relative proportion). It seems plausible that the first stage is the establishment of an unstable extrachromosomal unit, followed by the acquisition of stability via integration.

In situ hybridization can be used to show that transfected cells have donor material integrated into the resident chromosomes. Any given cell line has only a single site of integration; but the site is different in each line. Probably the selection of a site for integration is a random event; sometimes it is associated with a gross chromosomal rearrangement.

The sites at which exogenous material becomes integrated usually do not appear to have any sequence relationship to the transfected DNA. The integration event involves a nonhomologous recombination between the mass of added DNA and a random site in the genome. The recombination event may be provoked by the introduction of a double-strand break into the chromosomal DNA, possibly by the action of DNA repair enzymes that are induced by the free ends of the exogenous DNA. Integrants produced by the integration event are stable, and are therefore more useful than transient transfectants for expriments that rely on the expression of the transfected gene (for review see Pellicer, 1980).

An exciting development of transfection techniques is their application to introduce genes into animals. An animal that gains new genetic information from the addition of foreign DNA is described as transgenic. The approach of directly injecting DNA can be used with mouse eggs, as shown in Figure 17.31. Plasmids carrying the gene of interest are injected into the germinal vesicle (nucleus) of the oocyte or into the pronucleus of the fertilized egg. The egg is implanted into a pseudopregnant mouse. After birth, the recipient mouse can be examined to see whether it has gained the foreign DNA, and, if so, whether it is expressed. (For a description of the technique see 100.5 Transgenic mice: Expression of Foreign Genes in Animals.)

The first questions we ask about any transgenic animal are how many copies it has of the foreign material, where these copies are located, and whether they are present in the germline and inherited in a Mendelian manner. The usual result of such experiments is that a reasonable minority (say ~15%) of the injected mice carry the transfected sequence. Usually, multiple copies of the plasmid appear to have been integrated in a tandem array into a single chromosomal site. The number of copies varies from 1 V150. They are inherited by the progeny of the injected mouse as expected of a Mendelian locus.

An important issue that can be addressed by experiments with transgenic animals concerns the independence of genes and the effects of the region within which they reside. If we take a gene, including the flanking sequences that contain its known regulatory elements, can it be expressed independently of its location in the genome? In other words, do the regulatory elements function independently, or is gene expression in addition controlled by other effects, for example, location in an appropriate chromosomal domain?

Are transfected genes expressed with the proper developmental specificity? The general rule now appears to be that there is a reasonable facsimile of proper control: the transfected genes are generally expressed in appropriate cells and at the usual time. There are exceptions, however, in which a transfected gene is expressed in an inappropriate tissue.

In the progeny of the injected mice, expression of the donor gene is extremely variable; it may be extinguished entirely, reduced somewhat, or even increased. Even in the original parents, the level of gene expression does not correlate with the number of tandemly integrated genes. Probably only some of the genes are active. In addition to the question of how many of the gene copies are capable of being activated, a parameter influencing regulation could be the relationship between the gene number and the regulatory proteins: a large number of promoters could dilute out any regulator proteins present in limiting amounts.

What is responsible for the variation in gene expression? One possibility that has often been discussed for transfected genes (and which applies also to integrated retroviral genomes) is that the site of integration is important. Perhaps a gene is expressed if it integrates within an active domain, but not if it integrates in another area of chromatin. Another possibility is the occurrence of epigenetic modification; for example, changes in the pattern of methylation might be responsible for changes in activity. Alternatively, the genes that happened to be active in the parents may have been deleted or amplified in the progeny.

A particularly striking example of the effects of an injected gene is provided by a strain of transgenic mice derived from eggs injected with a fusion consisting of the MT promoter linked to the rat growth hormone structural gene. Growth hormone levels in some of the transgenic mice were several hundred times greater than normal. The mice grew to nearly twice the size of normal mice, as can be seen from Figure 17.32.

The introduction of oncogene sequences can lead to tumor formation. Transgenic mice containing the SV40 early coding region and regulatory elements express the viral genes for large T and small t antigens only in some tissues, most often brain, thymus, and kidney. (The T/t antigens are alternatively spliced proteins coded by the early region of the virus; they have the ability to transform cultured cells to a tumorigenic phenotype; see 28 Oncogenes and cancer.) The transgenic mice usually die before reaching 6 months, as the result of developing tumors in the brain; sometimes tumors are found also in thymus and kidney. Different oncogenes may be used to generate mice developing various cancers, thus making possible a range of model systems. For example, introduction of the myc gene under control of an active promoter causes the appearance of adenocarcinomas and other tumors.

Can defective genes be replaced by functional genes in the germline using transgenic techniques? One successful case is represented by a cure of the defect in the hypogonadal mouse. The hpg mouse has a deletion that removes the distal part of the gene coding for the polyprotein precursor to GnRH (gonadotropin-releasing hormone) and GnRH-associated peptide (GAP). As a result, the mouse is infertile.

When an intact hpg gene is introduced into the mouse by transgenic techniques, it is expressed in the appropriate tissues. Figure 17.33 summarizes experiments to introduce a transgene into hpg/hpg homozygous mutant mice. The resulting mice are normal. This provides a striking demonstration that expression of a transgene under normal regulatory control can be indistinguishable from the behavior of the normal allele.

Impediments to using such techniques to cure genetic defects at present are that the transgene must be introduced into the germline of the preceding generation, the ability to express a transgene is not predictable, and an adequate level of expression of a transgene may be obtained in only a small minority of the transgenic animals. Also, the large number of transgenes that may be introduced into the germline, and their erratic expression, could pose problems for the animal in cases in which over-expression of the transgene was harmful.

In the hpg murine experiments, for example, only 2 out of 250 eggs mice injected with intact hpg genes gave rise to transgenic mice. Each transgenic animal contained >20 copies of the transgene. Only 20 of the 48 offspring of the transgenic mice retained the transgenic trait. When inherited by their offspring, however, the transgene(s) could substitute for the lack of endogenous hpg genes. Gene replacement via a transgene is therefore effective only under restricted conditions.

The disadvantage of direct injection of DNA is the introduction of multiple copies, their variable expression, and often difficulty in cloning the insertion site because sequence rearrangements may have been generated in the host DNA. An alternative procedure is to use a retroviral vector carrying the donor gene. A single proviral copy inserts at a chromosomal site, without inducing any rearrangement of the host DNA. It is possible also to treat cells at different stages of development, and thus to target a particular somatic tissue; however, it is difficult to infect germ cells.

A powerful technique for making transgenic mice takes advantage of embryonic stem (ES) cells, which are derived from the mouse blastocyst (an early stage of development, which precedes implantation of the egg in the uterus). Figure 17.34 illustrates the principles of this technique.

ES cells are transfected with DNA in the usual way (most often by microinjection or electroporation). By using a donor that carries an additional sequence such as a drug resistance marker or some particular enzyme, it is possible to select ES cells that have obtained an integrated transgene carrying any particular donor trait. An alternative is to use PCR technology to assay the transfected ES cells for successful integration of the donor DNA. By such means, a population of ES cells is obtained in which there is a high proportion carrying the marker.

These ES cells are then injected into a recipient blastocyst. The ability of the ES cells to participate in normal development of the blastocyst forms the basis of the technique. The blastocyst is implanted into a foster mother, and in due course develops into a chimeric mouse. Some of the tissues of the chimeric mice will be derived from the cells of the recipient blastocyst; other tissues will be derived from the injected ES cells. The proportion of tissues in the adult mouse that are derived from cells in the recipient blastocyst and from injected ES cells varies widely in individual progeny; if a visible marker (such as coat color gene) is used, areas of tissue representing each type of cell can be seen.

To determine whether the ES cells contributed to the germline, the chimeric mouse is crossed with a mouse that lacks the donor trait. Any progeny that have the trait must be derived from germ cells that have descended from the injected ES cells. By this means, an entire mouse has been generated from an original ES cell (for review see Jaenisch, 1988)!

A further development of these techniques makes it possible to obtain homologous recombinants. A particular use of homologous recombination is to disrupt endogenous genes, as illustrated in Figure 17.35. A wild-type gene is modified by interrupting an exon with a marker sequence; most often the neo gene that confers resistance to the drug G418 is used. Also, another marker is added on one side of the gene; for example, the TK gene of the herpes virus. When this DNA is introduced into an ES cell, it may be inserted into the genome by either nonhomologous or homologous recombination. A nonhomologous recombination inserts the whole unit, including the flanking TK sequence. But a homologous recombination requires two exchanges, as a result of which the flanking TK sequence is lost. Cells in which a homologous recombination has occurred can therefore be selected by the gain of neo resistance and absence of TK activity (which can be selected because TK causes sensitivity to the drug gancyclovir). If it is not convenient to use a selectable marker such as TK, cells can simply be screened by PCR assays for the absence of flanking DNA. The frequency of homologous recombination is ~10 ^V7, and probably represents <1% of all recombination events (for review see Capecchi, 1989).

The presence of the neo gene in an exon disrupts transcription, and thereby creates a null allele. A particular target gene can therefore be "knocked out" by this means; and once a mouse with one null allele has been obtained, it can be bred to generate the homozygote. This is a powerful technique for investigating whether a particular gene is essential, and what functions in the animal are perturbed by its loss.

A sophisticated method for introducing new DNA sequences has been developed with D. melanogaster by taking advantage of the P element. The protocol is illustrated in Figure 17.36. A defective P element carrying the gene of interest is injected together with an intact P element into preblastoderm embryos. The intact P element provides a transposase that recognizes not only its own ends but also those of the defective element. As a result, either or both elements may be inserted into the genome (Spradling and Rubin, 1982).

Only the sequences between the ends of the P DNA are inserted; the sequences on either side are not part of the transposable element. An advantage of this technique is that only a single element is inserted in any one event, so the transgenic flies usually carry only one copy of the foreign gene, a great aid in analyzing its behavior.

Several genes that have been introduced in this way all show the same behavior. They are expressed only in the appropriate tissues and at the proper times during development, irrespective of the site of integration. So in D. melanogaster, all the information needed to regulate gene expression may be contained within the gene locus itself, and can be relatively impervious to external influence.

With these experiments, we see the possibility of extending from cultured cells to animals the option of examining the regulatory features. The ability to introduce DNA into the genotype allows us to make changes in it, to add new genes that have had particular modifications introduced in vitro, or to inactivate existing genes. So it may become possible to delineate the features responsible for tissue-specific gene expression. Ultimately it may be possible to replace defective genes in the genotype in a targeted manner.