TRANSGENIC MOUSE WITH ONE COPY OF TARGET GENE REPLACED BY ALTERED GENE IN GERM LINE SOMATIC CELLS OF OFFSPRING TESTED FOR PRESENCE OF ALTERED GENE, AND SELECTED MICE BRED TO TEST FOR GENE IN GERM-LINE CELLS BIRTH MATE AND HARVEST EARLY EMBRYOS HYBRID EARLY EMBRYO PARTLY FORMED FROM ES CELLS INTRODUCE HYBRID EARLY EMBRYO INTO PSEUDOPREGNANT MOUSE isolated early embryo in culture altered version of target gene constructed by genetic engineering INTRODUCE A DNA FRAGMENT CONTAINING ALTERED GENE INTO MANY CELLS TEST FOR THE RARE COLONY IN WHICH THE DNA FRAGMENT HAS REPLACED ONE COPY OF THE NORMAL GENE LET EACH CELL PROLIFERATE TO FORM A COLONY ES cells with one copy of target gene replaced by mutant gene INJECT ES CELLS INTO EARLY EMBRYO
The ability to prepare transgenic mice lacking a known normal gene has been a major advance, and the technique has been used to determine the functions of many mouse genes (Figure 8–54). If the gene functions in early development, a knockout mouse will usually die before it reaches adulthood. These lethal defects can be carefully analyzed to help determine the function of the missing gene. As described in Chapter 5, an especially useful type of transgenic animal takes advantage of a site-specific recombination system to excise—and thus disable— the target gene in a particular place or at a particular time (see Figure 5–66). In this case, the target gene in embryonic stem (ES) cells is replaced by a fully functional version of the gene that is flanked by a pair of the short DNA sequences, called lox sites, that are recognized by the Cre recombinase protein. The transgenic mice that result are phenotypically normal. They are then mated with transgenic mice that express the Cre recombinase gene under the control of an inducible promoter. In
Figure 8–53 Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured ES (embryonic stem) cells. These cells are described in detail in Chapter 22. Only a few ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. These cells can be identified by PCR and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells (indicated by orange) that carry the altered gene. Some of these mice will also contain germ-line cells that contain the altered gene; when bred with a normal mouse, some of the progeny of these mice will contain one copy of the altered gene in all of their cells.
The mice with the transgene in their germ line are then bred to produce both a male and a female animal, each heterozygous for the gene replacement (that is, they have one normal and one mutant copy of the gene). When these two mice are mated (not shown), one-fourth of their progeny will be homozygous for the altered gene.
the specific cells or tissues in which Cre is switched on, it catalyzes recombination between the lox sequences—excising a target gene and eliminating its activity (see Figure 22–5).
The Bacterial CRISPR System Has Been Adapted to Edit Genomes in a Wide Variety of Species
One of the difficulties in making transgenic mice by the procedure just described is that the introduced DNA molecule (bearing the experimentally altered gene) often inserts at random in the genome, and many ES cells must therefore be screened individually to find one that has the “correct” gene replacement.