Pcr

kanMX

Disruption construct

Diploid cell

Four haploid spores kanMX

Diploid cell

Transform diploid cells with disruption construct

Homologous recombination

Select for G-418 resistance

Transform diploid cells with disruption construct

Four haploid spores

Homologous recombination

Select for G-418 resistance

If the disrupted gene is essential, these spores will be nonviable

If the disrupted gene is essential, these spores will be nonviable

▲ EXPERIMENTAL FIGURE 9-37 Homologous recombination with transfected disruption constructs can inactivate specific target genes in yeast. (a) A suitable construct for disrupting a target gene can be prepared by the PCR. The two primers designed for this purpose each contain a sequence of about 20 nucleotides (nt) that is homologous to one end of the target yeast gene as well as sequences needed to amplify a segment of DNA carrying a selectable marker gene such as kanMX, which confers resistance to G-418. (b) When recipient diploid Saccharomyces cells are transformed with the gene disruption construct, homologous recombination between the ends of the construct and the corresponding chromosomal sequences will integrate the kanMX gene into the chromosome, replacing the target gene sequence. The recombinant diploid cells will grow on a medium containing G-418, whereas nontransformed cells will not. If the target gene is essential for viability, half the haploid spores that form after sporulation of recombinant diploid cells will be nonviable.

which like neor confers resistance to G-418) flanked by about 20 base pairs that match the ends of the target yeast gene. Transformed diploid yeast cells in which one of the two copies of the target endogenous gene has been replaced by the disruption construct are identified by their resistance to G-418 or other selectable phenotype. These heterozygous diploid yeast cells generally grow normally regardless of the function of the target gene, but half the haploid spores derived from these cells will carry only the disrupted allele (Figure 9-37b). If a gene is essential for viability, then spores carrying a disrupted allele will not survive.

Disruption of yeast genes by this method is proving particularly useful in assessing the role of proteins identified by ORF analysis of the entire genomic DNA sequence. A large consortium of scientists has replaced each of the approximately 6000 genes identified by ORF analysis with the kanMX disruption construct and determined which gene disruptions lead to non-viable haploid spores. These analyses have shown that about 4500 of the 6000 yeast genes are not required for viability, an unexpectedly large number of apparently nonessential genes. In some cases, disruption of a particular gene may give rise to subtle defects that do not compromise the viability of yeast cells growing under laboratory conditions. Alternatively, cells carrying a disrupted gene may be viable because of operation of backup or compensatory pathways. To investigate this possibility, yeast geneticists currently are searching for synthetic lethal mutations that might reveal nonessential genes with redundant functions (see Figure 9-9c).

Transcription of Genes Ligated to a Regulated Promoter Can Be Controlled Experimentally

Although disruption of an essential gene required for cell growth will yield nonviable spores, this method provides little information about what the encoded protein actually does in cells. To learn more about how a specific gene contributes to cell growth and viability, investigators must be able to selectively inactivate the gene in a population of growing cells. One method for doing this employs a regulated promoter to selectively shut off transcription of an essential gene.

A useful promoter for this purpose is the yeast GAL1 promoter, which is active in cells grown on galactose but completely inactive in cells grown on glucose. In this approach, the coding sequence of an essential gene (X) ligated to the GAL1 promoter is inserted into a yeast shuttle vector (see Figure 9-19a). The recombinant vector then is introduced into haploid yeast cells in which gene X has been disrupted. Haploid cells that are transformed will grow on galactose medium, since the normal copy of gene X on the vector is expressed in the presence of galactose. When the cells are transferred to a glucose-containing medium, gene X no longer is transcribed; as the cells divide, the amount of the encoded protein X gradually declines, eventually reaching a state of depletion that mimics a complete loss-of-function mutation. The observed changes in the phenotype of these cells after the shift to glucose medium may suggest which cell processes depend on the protein encoded by the essential gene X.

In an early application of this method, researchers explored the function of cytosolic Hsc70 genes in yeast. Hap-loid cells with a disruption in all four redundant Hsc70 genes were nonviable, unless the cells carried a vector containing a copy of the Hsc70 gene that could be expressed from the GAL1 promoter on galactose medium. On transfer to glucose, the vector-carrying cells eventually stopped growing because of insufficient Hsc70 activity. Careful examination of these dying cells revealed that their secretory proteins could no longer enter the endoplasmic reticulum (ER). This study provided the first evidence for the unexpected role of Hsc70 protein in translocation of secretory proteins into the ER, a process examined in detail in Chapter 16.

Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice

Many of the methods for disrupting genes in yeast can be applied to genes of higher eukaryotes. These genes can be introduced into the germ line via homologous recombination to produce animals with a gene knockout, or simply "knockout." Knockout mice in which a specific gene is disrupted are a powerful experimental system for studying mammalian development, behavior, and physiology. They also are useful in studying the molecular basis of certain human genetic diseases.

Gene-targeted knockout mice are generated by a two-stage procedure. In the first stage, a DNA construct contain-

► EXPERIMENTAL FIGURE 9-38 Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice. (a) When exogenous DNA is introduced into embryonic stem (ES) cells, random insertion via nonhomologous recombination occurs much more frequently than gene-targeted insertion via homologous recombination. Recombinant cells in which one allele of gene X (orange and white) is disrupted can be obtained by using a recombinant vector that carries gene X disrupted with ned (green), which confers resistance to G-418, and, outside the region of homology, t^HSV (yellow), the thymidine kinase gene from herpes simplex virus. The viral thymidine kinase, unlike the endogenous mouse enzyme, can convert the nucleotide analog ganciclovir into the monophosphate form; this is then modified to the triphosphate form, which inhibits cellular DNA replication in ES cells. Thus ganciclovir is cytotoxic for recombinant ES cells carrying the tkHSV gene. Nonhomologous insertion includes the tkHSV gene, whereas homologous insertion does not; therefore, only cells with nonhomologous insertion are sensitive to ganciclovir. (b) Recombinant cells are selected by treatment with G-418, since cells that fail to pick up DNA or integrate it into their genome are sensitive to this cytotoxic compound. The surviving recombinant cells are treated with ganciclovir. Only cells with a targeted disruption in gene X, and therefore lacking the tkHSV gene, will survive. [See S. L. Mansour et al., 1988, Nature 336:348.]

ing a disrupted allele of a particular target gene is introduced into embryonic stem (ES) cells. These cells, which are derived from the blastocyst, can be grown in culture through many generations (see Figure 22-3). In a small fraction of trans-fected cells, the introduced DNA undergoes homologous recombination with the target gene, although recombination at nonhomologous chromosomal sites occurs much more frequently. To select for cells in which homologous gene-targeted insertion occurs, the recombinant DNA construct introduced into ES cells needs to include two selectable marker genes (Figure 9-38). One of these genes (neor), which

(a) Formation of ES cells carrying a knockout mutation neor tkHSV

Gene X replacement construct

Homologous recombinatior

Homologous recombinatior

ES cells

ES-cell DNA

Gene X

Gene-targeted insertion

ES cells

ES cells

ES cells

Nonhomologous recombination

Nonhomologous recombination

ES-cell DNA

Gene X

Other genes

Gene-targeted insertion

Random insertion

Mutation in gene X

Cells are resistant to G-418 and ganciclovir

No mutation in gene X

Cells are resistant to G-418 but sensitive to ganciclovir

(b) Positive and negative selection of recombinant ES cells

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