Overview of Gene Targeting by Homologous UNIT 4.29 Recombination The analysis of mutant organisms and cell tion of the mutation present in the targeting lines has been important in determining the construct into the target gene. Once identified, function of specific proteins. Until recently, mutant ES cell clones can be microinjected into mutants were produced by mutagenesis fol- a normal blastocyst in order to produce a lowed by selection for a particular phenotypic chimeric mouse. Because many ES cell lines change. Recent technological advances in gene retain the ability to differentiate into every cell targeting by homologous recombination in type present in the mouse, the chimera can have mammalian systems enable the production of tissues, including the germ line, with contribu- mutants in any desired gene (Mansour, 1990; tion from both the normal blastocyst and the Robertson, 1991; Zimmer, 1992). This technol- mutant ES cells. Breeding germ-line chimeras ogy can be used to produce mutant mouse yields animals that are heterozygous for the strains and mutant cell lines. Because most mutation introduced into the ES cell, and that mammalian cells are diploid, they contain two can be interbred to produce homozygous mu- copies, or alleles, of each gene encoded on an tant mice. autosomal (nonsex) chromosome. In most Homologous recombination can also be cases, both alleles must be inactivated to pro- used to produce homozygous mutant cell lines. duce a discernible phenotypic change in a mu- Previously, inactivation of both alleles of a gene tant. The conversion from heterozygosity to required two rounds of homologous recombi- homozygosity is accomplished by breeding in nation and selection (te Riele et al., 1990; Cruz the case of mouse strains and by direct selection et al., 1991; Mortensen et al., 1991). Now, in cell lines. however, inactivation of both alleles of many Bacteriophage recombinases such as Cre genes requires only a single round of homolo- and its recognition sequence, loxP, have also gous recombination using a single targeting allowed spatial control of knockouts. Another construct (Mortensen et al., 1992). The ho- recombinase system, the yeast Flp/FRT system, mozygous mutant cells can then be analyzed can also be used (Fiering et al., 1993, 1999). for phenotypic changes to determine the func- The control can function along actual spatial tion of the gene. coordinates when a viral gene transfer system is used, or in a cell type- or tissue-specific ANATOMY OF TARGETING fashion when restricted promoters are em- CONSTRUCTS ployed. Adding temporal regulation of Cre, Two basic configurations of constructs are such as that achievable with the tetracycline- used for homologous recombination: insertion regulatable expression system (see CPMB UNIT constructs and replacement constructs (Fig. 16.21 and APPENDIX 1A in this manual), allows 4.29.1). Each can be used for different pur- temporal control as well. poses in specific situations, as discussed be- To produce a mutant mouse strain by ho- low. The insertion construct contains a region mologous recombination, two major elements of homology to the target gene cloned as a are needed. An embryonic stem (ES) cell line single continuous sequence, and is linearized capable of contributing to the germ line, and a by cleavage of a unique restriction site within targeting construct containing target-gene se- the region of homology. Homologous recom- quences with the desired mutation. Maintain- bination introduces the insertion construct se- ing ES cells in their undifferentiated state is a quences into the homologous site of the target major task during gene targeting (see CPMB UNIT gene, interrupting normal target-gene struc- 23.3 and APPENDIX 1A in this manual). This usually ture by adding sequences. As a result, the is accomplished by growing cells on a layer of normal gene can be regenerated from the mu- feeder cells (see CPMB UNIT 23.2 and APPENDIX 1A tated target gene by an intrachromosomal re- in this manual). The targeting construct is then combination event. transfected into cultured ES cells (see UNIT 4.30). The replacement construct is the second, ES cell lines are derived from the inner cell more commonly used construct. It contains two mass of a blastocyst-stage embryo. Homolo- regions of homology to the target gene located gous recombination occurs in a small number on either side of a mutation (usually a positive Gene Cloning, of the transfected cells, resulting in introduc- selectable marker; see below). Homologous Expression, and Mutagenesis Contributed by Richard Mortensen 4.29.1 Current Protocols in Neuroscience (2002) 4.29.1-4.29.10 Copyright © 2002 by John Wiley & Sons, Inc. Supplement 21 insertion construct replacement construct neo neo ∗ ∗ ∗ ∗ 2 3 2 3 1 2 3 4 1 2 3 4 neo neo ∗ ∗ ∗ ∗ 1 2 3 2 3 4 1 2 3 4 Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene vector Figure 4.29.1 Two configurations of constructs used for homologous recombination. Numbers indicate target-gene sequences in the genome. An asterisk indicates homologous target-gene sequences in the construct. Replacement constructs substitute their sequences (2*, neo, and 3*) for the endogenous target-gene sequences (2 and 3). Insertion constructs add their sequences (2*, neo, and 3*) to the endogenous target gene, resulting in tandem duplication and disruption of the normal gene structure. recombination proceeds by a double cross-over Positive-Negative Selection event that replaces the target-gene sequences The most commonly used method for elimi- with the replacement-construct sequences. Be- nating cells in which the construct integrated cause no duplication of sequences occurs, the into the genome randomly, thus further enrich- normal gene cannot be regenerated. ing for homologous recombinants, is known as positive-negative selection. It is only applicable METHODS OF ENRICHMENT FOR to replacement constructs (Fig. 4.29.2; Man- HOMOLOGOUS RECOMBINANTS sour et al., 1988). In these constructs, a negative selectable marker (e.g., herpes simplex virus Positive Selection by Drug-Resistance thymidine kinase, HSV-TK) is included outside Gene the region of homology to the target gene. In Nearly all constructs used for homologous the presence of the TK gene, the cells are recombination rely on the positive selection sensitive to acyclovir and its analogs (e.g., of a drug-resistance gene (e.g., neomycin or gancyclovir, GANC). The HSV-TK enzyme neo) that is also used to interrupt and mutate activates these drugs, resulting in their incor- the target gene. When either insertion or re- poration into growing DNA, causing chain ter- placement constructs are linearized, the drug- mination and cell death. During homologous resistance gene is flanked by two regions of recombination, sequences outside the regions homology to the target gene. Selection of the of homology to the target gene are lost due to cells using drugs (e.g., G418) eliminates the crossing over. In contrast, during random inte- great majority of transformants that have not gration all sequences in the construct tend to be stably incorporated the construct (see CPMB UNIT retained because recombination usually occurs 9.5 and APPENDIX 1A in this manual). However, in at the ends of the construct. The presence of the many of the surviving clones the construct has TK gene can be selected against by growing the incorporated into the genome not by homolo- cells in gancyclovir; the homologous recombi- gous recombination but rather through random nants will be G418-resistant and gancyclovir- Overview of Gene integration. Therefore, methods to enrich for resistant, whereas clones in which the construct Targeting by Homologous homologous recombinant clones have been de- integrated randomly will be G418-resistant and Recombination veloped. gancyclovir-sensitive. In some cases, TK is 4.29.2 Supplement 21 Current Protocols in Neuroscience homologous recombination random integration neo TK neo TK ∗ ∗ ∗ ∗ 2 3 2 3 1 2 3 4 neo neo TK ∗ ∗ ∗ ∗ 1 2 3 4 2 3 cell phenotype: G418R G418R GANCR GANCS Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene negative selectable marker vector promoter Figure 4.29.2 Enrichment for homologous recombinants by positive-negative selection using the TK gene. Homologous recombination involving cross-overs on either side of the neo gene results in loss of the TK gene. Random integration tends to preserve the TK gene. The presence of TK can be selected against because any cell expressing the gene will be killed by gancyclovir (GANC). Although both homologous recombinants and clones in which the construct integrated randomly are G418-resistant, only homologous recombinants are gancyclovir-resistant. The construct is shown linearized so that the plasmid vector sequences remain attached to the TK gene. This configuration helps preserve the integrity of the TK gene. The superscript R denotes resistance and the superscript S denotes sensitivity. inactivated without homologous recombina- recombination occurs, a fusion protein is pro- tion; thus, the gancyclovir-resistant clones duced, driven by the endogenous target-gene must be screened to identify the true homolo- promoter. In contrast, when random integration gous recombinants. Other markers that are le- occurs, the selectable-marker protein is not thal to cells have also been used instead of TK usually produced. Therefore, homologous re- and gancyclovir (e.g., diphtheria toxin; Yagi et combinants are G418-resistant, whereas cells al., 1990). in which the construct integrated randomly are G418-sensitive. Constructs containing a pro- Endogenous Promoters moterless selectable marker can be constructed Constructs that rely on an endogenous pro- in either replacement or insertion structure and moter to express the positive selectable marker can result in dramatic enrichment for homolo- can also give enrichment of homologous re- gous recombinants. combinants (Fig. 4.29.3), but can only be used if the gene of interest is expressed in the cell TYPES OF MUTATIONS line.