The analysis of mutant organisms and cell lines has

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1 646 Double Knockouts Production of Mutant Cell Lines in Cardiovascular Research Richard M. Mortensen Double knockouts by homologous recombination is a method Tor producing cell lines with an inactivating mutation in any desired gene. The biochemical analysis of genetically altered cell lines has been important in determining the function of specific proteins. Until recently, mutant cell lines have been produced by random mutagenesis and then selection for a particular phenotypic change. Recent technological advances in gene targeting by homologous recombination now enable the production of mutants in any desired gene. Diploid cells contain two copies or alleles of each gene encoded on an autosome (nonsex) chromosome. In most cases, both alleles must be inactivated to produce a phenotypic change in a mutant cell line, hence the term "double knockout." We and others have described the production of mutationally altered cell lines by inactivating both alleles by the production of two targeting vectors, two separate homologous recombination events, and selection. A simpler procedure, involving considerably less effort and time, has been used to inactivate several or-subunits of G proteins and other genes. This method facilitates the inactlvation of more than one gene in a single cell line. (Hypertension. 1993;22: ) KEY WORDS genetics cell line recombination, genetic mutation The analysis of mutant organisms and cell lines has been important in determining the function of specific proteins. Until recently, mutants have been produced by random mutagenesis and then selection for a particular phenotypic change. Recent technological advances in gene targeting by homologous recombination in mammalian systems now enable the production of mutants in any desired gene. 10 This technology can be used to produce mutant mouse strains and mutant cell lines. Diploid cells contain two copies or alleles of each gene encoded on an autosome or nonsex chromosome. In most cases, both alleles must be inactivated to produce a phenotypic change in a mutant. The conversion to homozygous mutations is accomplished by breeding in the case of mouse strains and by direct means for cell lines. To produce a mutant mouse strain, the desired mutation is first introduced into cloned DNA sequences. This targeting construct is then transfected into a cultured embryonic stem (ES) cell line. These cell lines are derived from the inner cell mass of normal blastocysts and maintain the ability to differentiate into every cell type. Homologous recombination will occur in a small number of these cells, resulting in introduction of the mutation into the genome. Once identified, Received March 17, 1993; accepted in revised form June 21, From Brigham and Women's Hospital, Harvard Medical School, Boston, Mass. Presented as a part of the Sunday Afternoon Program "Manipulating Genes to Understand Cardiovascular Diseases: Principles, Methodologies, and Applications" at the American Heart Association's 65th Scientific Sessions, New Orleans, La, November 15, Reprint requests to Richard M. Mortensen, MD, PhD, Endocrine-Hypertension Division, 221 Longwood Ave, Boston, MA these mutant clones can be microinjected into a normal blastocyst to produce a chimera. A chimera can have tissues, including the germ line, with contribution from the normal blastocyst and from the injected ES cells. Breeding these germ-line chimeras yields animals heterozygous for the mutation introduced into the ES cell. Heterozygotes can be bred to produce homozygotes. Homologous recombination can also be applied to produce mutant cultured cell lines. Although previously inactivation of both copies of a gene required two rounds of homologous recombination and selection, 46 inactivation of many genes now only requires the production of a single targeting construct. 7 These mutants can be analyzed for phenotypic changes to determine the function of the gene. This method facilitates the inactivation of more than one gene in a single cell line. Gene Targeting of the First Allele There are two basic configurations of constructs for homologous recombination, termed insertion and replacement constructs (Fig 1A). In insertion constructs, the sequences in the targeting vector are introduced into the homologous site, interrupting the normal structure of the gene. The region of homology to the target gene is cloned as a single continuous sequence, and the construct is linearized by cleavage at a unique restriction site in the region of homology. Homologous recombination adds sequences to the target gene. As a result, the normal gene can be regenerated by an intrachromosomal recombination event. The second type of construct is more commonly used and is termed a replacement construct. There are two regions homologous to the target gene on either side of a mutation (usually a positive selectable marker, see below). Homologous recombination proceeds by a double crossover event that results in the replacement of the endogenous sequences with the construct se-

2 Mortensen Homologous Recombination 647 A CONSTRUCT TYPES INSERTION CONSTRUCT REPLACEMENT CONSTRUCT 2* 3* x 4 -» 1 2 I 3 I 1 2 3* 2' 3 B ENRICHMENT BY POSITIVE-NEGATIVE SELECTION HOMOLOGOUS RECOMBINATION 2' 3* RANDOM INSERTION TK 2* 3* CELL PHENOTYPE: G418R GANCS Homologous sequences construct and genomtc Non-homologous sequences positive selectable marker genomtc sequences negative selectable marker E2 vector sequences FIG 1. A: Diagrams show two configurations for homologous recombination. Homologous sequences in the construct are indicated by an asterisk. Replacement constructs substitute their sequences (exon 2* neo, exon 3*) for the endogenous sequences (cxons 2 and 3). Insertion constructs add their sequences to the genome, resulting in tandem duplication and disruption of the normal gene structure. Insertion constructs could also incorporate interruption of an exon by neo. B: Diagram shows enrichment by positive-negative selection using the herpes simplex virus thymidine kinase gene (HSV- TK). Crossover on either side of the neo gene results in bss of the TK if homologous recombination occurs. Random insertion tends to preserve the TK. Presence of the TK gene can be selected against because any cell expressing the gene will be killed by gancyclovir (GANC). The construct is shown linearized so that the plasmid vector sequences are attached to the TKgene. This configuration helps preserve the integrity of the TKgene. Superscript R denotes resistance to the antibiotic; S denotes sensitivity. quences. No duplication of sequences occurs, so the normal gene cannot be regenerated. Nearly all constructs for homologous recombination rely on the positive selection of a drug resistance gene (eg, neo) that is usually also used to produce the interruption in the gene. When either an insertion or replacement construct is linearized, the positive selectable marker is flanked by two regions homologous to the target gene. These regions of genomic sequence provide the substrates for homologous recombination. Generally, the homology regions should be greater than 1 kb on each side of the neo gene, with a total homology of 6 kb or greater. The degree of homology between the construct and the target genome can have a dramatic effect on the rate of homologous recombination in two ways. First, the DNA used to construct the targeting vector must be from the same species as the cell in which the mutation is to be introduced. Homologous recombination requires stretches of exact homology. Because different animal strains may differ just as

3 648 Hypertension Vol 22, No 4 October 1993 Construct Homologous rvcomunant Nail 81 kb TfSBl ±± FIG 2. Diagram shows structure of the targeting construct used to inactivate the a^ gene. The construct has approximately 7 kb of homology and the neo gene interrupting exon 6. Homologous recombination is detected by Southern analysis by digestion ofgenomic DNA with Nsi / and hybridization with a probe (BamHI-BamHI) outside the region of homology with the construct (size of predicted hybridizing fragments shown as solid bars). Adapted from Mortensen et al. 7 individual outbred animals differ, there may be a mismatch on average every 500 bp; therefore, it is best if the homology segments are derived from DNA that is isogenic with the target DNA (ie, from the same strain of animal or made from the cell line directly). The nonisogenic mismatch is sufficient to dramatically decrease the rate of homologous recombination. 89 However, constructs derived from nonisogenic DNA often work well. Secondly, the longer the homologous regions, the higher the rates of recombination (within limits). The exact length at which creating longer constructs will not increase recombination rates is controversial but may be up to 15 kb. 810 Furthermore, fidelity of recombination can be lower if the length of homology is shorter than 1 kb on a side. 11 For the a^ gene, the construct was devised as a replacement vector using positive-negative selection (illustrated in Fig IB and explained below). The neo was inserted into the BamHl site in exon 6, and the a$ gene was interrupted at the Neo I site at the initiating ATG codon in exon 1 (Fig 2). 5 The neo coding sequence was expressed using a promoter active in ES cells. Neither of these constructs was isogenic with the target DNA, because they were derived from a BALB/c genomic library and the ES cells are derived from the 129Sv murine strain. The construct is introduced into ES cells by electroporation. Selection of the cells in G418 eliminates the great majority of clones that do not stably incorporate the construct. However, many of the clones will incorporate the construct through random integration in the genome. To enrich for homologous recombinant colonies, the herpes simplex virus thymidine kinase gene (HSV-TK) is included outside the regions of homology. Cells lacking the TK gene can be selected by treatment of a cell culture with gancyclovir. 12 This method for further enrichment of homologous recombinants is termed positive-negative selection (Fig IB). It is applicable to replacement constructs only. The presence of this thymidine kinase gene makes cells sensitive to acyclovir and its analogues such as gancyclovir. HSV-TK enzyme can activate these drugs, which are then incorporated into growing DNA during synthesis, causing chain termination and death of the cell. Sequences outside the regions of homology will be lost during homologous recombination because of crossover. However, all sequences in the construct will tend to be retained if random integration occurs, because recombination usually occurs at the ends of the construct. The presence of the HSV-TK gene can be selected against by gancyclovir. Therefore, homologous recombinants will be neo resistant and gancyclovir resistant, whereas random integration of the construct will give neo resistance but also gancyclovir sensitivity. In some cases, the TK will be inactivated without homologous recombination so that surviving clones must be screened for the true homologous recombinants. Other markers that are lethal to cells containing them have also been used, eg, diphtheria toxin. 13 The degree of enrichment varies but is generally fivefold to 10-fold (0,2, threefold to fivefold; a-,, 10-fold). The CCQ targeting vector was transfected into the cells, selected in G418 and gancyclovir, and the surviving colonies screened for homologous recombination by Southern analysis. Correct homologous recombination results in a change in the size of the hybridizing band using a probe homologous to a region not contained in the construct (Fig 2). The probe, for detection of homologous recombination by Southern analysis, can usually be obtained from sequences in the original genomic clone that were not used for the construct. With the use of a probe outside the sequences in the construct, only the endogenous gene and homologous recombinants will be detected, not the construct randomly integrated into the genome. The probe should detect no more than a few bands on a Southern blot of genomic DNA. Only clones that have undergone homologous recombination are analyzed further. Usually, no other recombination events have occurred in these clones. However, it is prudent to test for a second copy of neo to ensure that a random integration did not occur in the same clone, because this could cause insertional mutagenesis or the failure of selection of double knockouts using the single construct method (below). Double Construct Method The second allele can be inactivated by a second round of gene targeting (Fig 3, Double Construct Method). Because the cells already express resistance to G418, a second selectable marker must be used to interrupt the exon. The hygromycin B resistance gene (hyg) has been used either as a fusion protein* or driven by its own promoter. 5-6 The second round is identical to the first. The second targeting construct can replace either the normal allele or the allele that has already been inactivated by neo. Homozygous mutants are determined by Southern analysis and the presence of both the hyg- and neo-altered alleles. This method is time consuming in that it requires the creation of a second targeting construct, and the rate of homologous recombination with the second construct will be similar to the first. For genes that undergo homologous recombination at low rates, screening a second time would be labor intensive. Most importantly, there are a very

4 Mortensen FIRST TARGETING 649 DOUBLE CONSTRUCT METHOD SINGLE CONSTRUCT METHOD 1 CREATE CONSTRUCT n»o Homologous Recombination CREATE CONSTRUCT TK hyg TRANSFECT TK TRANSFECT SELECT HGHO418 \ Homologoua MquanoM Non-homotogou» taquano pootthro NwtiibtB motor I K f l l lltli HKl!! * vtdof Mquanc««promotor saquano* SELECT LOW 0418 QANC SELECT LOW HYQROUYCIN B QANC SCREEN SCREEN D n SCREEN 0418 n HXMOlygote HYGROMYCJN B R HonoiygoM FIG 3. Diagram shows scheme for production of homozygous mutants using two separate targeting constructs or a single construct. Resulting cell lines from the two construct method are resistant to both G418 and hygromycin B, whereas using a single construct the homozygous cell is resistant only to G418. TK, herpes simplex virus thymidine kinase gene (HSV-TK); GANC, gancyclovir; superscript R, resistance to the antibiotic. limited number of usable selectable markers so that the inactivation of both alleles of two or more genes will be difficult or impossible. This is particularly important when investigating the function of a family of genes that may have overlapping phenotypes, because more than one member of a family would need to be inactivated to observe the desired phenotype. Single Construct Method A simpler, more efficient method can be used to obtain homozygous knockout cells that are resistant to only a single antibiotic (Fig 3, Single Construct Method). This method has been applied using a neomycin resistance gene that contains a point mutation resulting in lower phosphotransferase activity.14 If the wild-type gene is used, it may result in cells that are resistant to antibiotics even at very high levels. Once a heterozygous targeted clone has been obtained, a homozygous cell line can be isolated from these heterozygous cells by selecting cells that are resistant to higher concentrations of G418 than the homozygous cells. Cells that have lost heterozygosity contain two interrupted alleles and therefore contain two neo genes, which make the cells more resistant to G418. The heterozygous cells are selected at higher concentrations of G418 (>1.0 mg/ ml). The cells are cultured for 7 to 10 days until single surviving colonies are detected. For some genes, higher G418 concentrations may be required, presumably depending on the relative expression of the neomycin resistance gene at different genomic sites. The relative resistance of the heterozygous cells to G418 will also depend on the promoter used to drive the expression of neo or on the integration site so that other clones may require more or less G418. If cells overgrow plates and no single colonies are obtained, the cells should be replated using higher G418 levels (some clones have required greater than 10 mg/ml). The surviving clones are screened by Southern blot analysis exactly as performed for the isolation of the heterozygous cell line except that the Southern blot of the DNA derived from a double knockout (homozygous) clone will now completely lack the band corresponding to the gene segment found in the normal parent cell. Examples of Southern analysis of two clones homozygous for a^ gene inactivation are shown in Fig 4 (lanes 3 and 4). The frequency of homozygous clones differs between heterozygous clones because loss of heterozygosity ap- Nsl *~ FIG 4. Southern analysis shows DNA isolated from embryonic stem cells: wild type (+/+), heterozygous for gene disruption with neo (+/-), and homozygous for the inactivated allele ( / ). Similar to previously published data.7

5 650 Hypertension Vol 22, No 4 October 1993 Loss of Heterozygoslty In Embryonic Stem Cell Lines [G418], mg/ml Gene (Parent Une) a a (CCE) Cell Une 17E10 18D3 No. Cells Plated* 2X10 4 2X10 4 1x10= 1X10 5 5x10" 5X10 5 5X10 5 No. of Colonies % Homozygote (No. Analyzed) 100% (21) 12% (26) 53% (17) 4% (28) 43% (23) No. of Colonies % Homozygote (No. Analyzed) 0%(2) ND 88% (17) a 0 (CC1.2) A TCR-a (CCE) 44% (9) 24% (54) 50% (2) Myhc-b (CC1.2) 76 7% (76) ND indicates not determined. Delta from Mortensen et al. 7 Efficiency of plating 40% to 80%. pears to be a random event. Several separately derived heterozygous clones should be expanded in culture and subjected to selection with higher levels of G418, because there appears to be some clone-to-clone variation in resistance to this drug. The frequency of homozygous clones may also be influenced by the time in culture before selection because the homozygous clones are continuously produced and tend to accumulate with time. Although the frequency of homozygous clones varies from 4% to 100%, it is often around 50% (Table). These cells retain their normal karyotype, and so this method may be useful for studying genes in development. The mechanism by which these homozygous cells are produced is unclear. Possibilities include chromosomal loss and duplication, nondisjunction, or even gene conversion. Previous descriptions of spontaneous production of homozygous clones have shown that the mechanism may vary with cell type Further definition of the mechanism in ES cells will require cells that are heterozygous at multiple loci. Inactivation of More Than One Gene in a Single Cell Line Because the homozygous cells are only resistant to G418, other selectable markers such as the hygromycin B resistance gene could be used in further homologous recombination targeting constructs. Other selectable markers might also be used to inactivate three or more genes as long as the presence of two copies can be selected over the presence of one copy. In this way, the functions of genes with overlapping phenotypes might be investigated. Uses of Mutant Cell Lines Once mutant cell lines have been produced, the cells can be analyzed to confirm inactivation of the gene and for phenotypic differences. Expression can be evaluated by Northern blot analyses of total RNA or Western blot analyses of proteins isolated from the cell line. The interruption of exon 6 in the eta gene leads to complete absence of detectable eta mrna by Northern analysis. 7 For these ES cells lacking functional eta or a^ genes, coupling of various receptors to different effectors can be analyzed provided that the responses are present in the wild-type cells. So far, no receptors known to couple through Oj have been identified in ES cells, 5 so two approaches can be taken. First, receptors can be heterologously expressed in the undifferentiated ES cells. Cell lines expressing the receptor would be developed in the wild-type and the null mutant cell background and tested for coupling to adenylyl cyclase, phospholipase C, phospholipase A2, mitogenesis, or other G protein-mediated signals. This method requires the identification of the desired response in the wild-type cell line. Alternatively, ES cells offer a unique opportunity because they are capable of differentiating into any cell type. ES cells have been shown to differentiate into a number of cell types that are of interest to investigators in cardiovascular research. Cardiac cells express cardiac-specific a and /3 cardiac myosin. 18 ' 19 They beat and possess the same K + channels identified in normal heart. Vascular channels have also been identified in differentiating embryoid bodies (which are masses of cells derived from ES cells that have undergone differentiation into an embryo-like structure) and can even contain blood elements. 20 Provided that the phenotype can be analyzed with a single or few cells, these differentiated cells can then be used to analyze the phenotype of the disrupted gene. ES cells can also differentiate within the entire organism by injection into normal blastocysts (homozygous mutant cells offer no technical advantage over heterozygous cells if the goal is to obtain a mutant mouse line through germ-line transmission). For the analysis of homozygous knockout cells in a developing chimeric embryo, the cells can be tagged by introducing a gene that has a histochemically detectable product (such as 0-galactosidase). The contribution of these tagged cells to tissues could then be determined by analyzing for the histochemical tag. Identified differentiated cells derived from the homozygous knockout ES cells could also be analyzed for altered functions at the single cell level. Once a particular phenotype has been shown to be dependent on the presence of a gene product, the system can be easily used to study the structure-function relations. For ctj genes, mutants can be reintroduced into the null cells, and there, function can be studied. This is particularly useful for studying the G proteineffector interactions.

6 Application of the Single Construct Method to Other Cell Lines The suitability of this technique for the production of types of cells other than ES cells that lack a particular gene is less certain. Because homologous recombination and spontaneous loss of heterozygosity are known to occur in other cell lines, these same methods may be used to produce cell lines lacking other functional genes. However, other types of immortalized cells (eg, lymphocytes or fibroblasts) may not allow the targeting vector to undergo homologous recombination at the same frequency as found in ES cells. Still, homologous recombination at usable rates has been reported in immortalized cell lines. 21 Furthermore, many other types of immortalized cells are polyploid or aneuploid, so that particular problems may arise for any one cell type. The method described here is a general one that can be used to produce a large variety of cell lines, each one of which will lack a particular gene. This approach cannot be used to produce double knockout cell lines for genes that are required for cell viability. This method has been successfully used to produce cells that lack aa.ao, o-cardiac myosin heavy chain, ^-cardiac myosin heavy chain, and T-cell o-receptor genes. We expect that future studies will lead to the production of many other such cell lines and that the characterization of these cell lines will lead to the definition of the role of different gene products in cell structure and function. Acknowledgment R.M.M. was supported by a Clinician Scientist Award from the American Heart Association. References 1. Mansour SL. Gene targeting in murine embryonic stem cells: introduction of specific alterations into the mammalian genome. Genet Anal Tech AppL 1990,7: Robertson EJ. Using embryonic stem cells to introduce mutations into the mouse germ line. Biol Reprod, 1991;44: Zimmer A. Manipulating the genome by homologous recombination in embryonic stem cells. Annu Rev NeuroscL 1992; 15: teriele H, Maandag ER, Clarke A, Hooper M, Berns A. Consecutive inactivation of both alleles of the pun-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature. 1990; 348: Mortensen Homologous Recombination Mortensen RM, Zubiaur M, Neer EJ, Seidman JG. Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles. Proc Natl Acad Sci USA. 1991;88: Cruz A, Coburn CM, Beverley SM. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci USA. 1991;88: Mortensen RM, Conner DA, Chao S, Geisterfer LA, Seidman JG. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell BioL 1992;12: Deng C, Capecchi MR. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell BioL 1992;12: teriele H, Maandag ER, Berns A. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci USA. 1992;89: Hasty P, Rivera PJ, Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell BioL 1991;11: Thomas KR, Deng C, Capecchi MR. High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol Cell BioL 1992;12: Mansour SL, Thomas KR, Capecchi MR. Disruption of the protooncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988;336: Yagi T, Ikawa Y, Yoshida K, Shigetani Y, Takeda N, Mabuchi I, Yamamoto T, Aizawa S. Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection. Proc Natl Acad Sci USA. 1990;87: Yenofsky RL, Fine M, Pellow JW. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci USA. 1990;87: Rajan TV, Moffat LF, Frankel WN. Rate and mechanism of generation of beta 2-microglobulin mutants from a heterozygous murine cell line. J ImmunoL 1990;145: Potter TA, Zeff RA, Frankel W, Rajan TV. Mitotic recombination between homologous chromosomes generates H-2 somatic cell variants in vitro. Proc Natl Acad Sci USA. 1987;84: Wasmuth JJ, Vock HL. Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids. Cell 1984;36: Sanchez A, Jones WK, Gulick J, Doetschman T, Robbins J. Myosin heavy chain gene expression in mouse embryoid bodies: an in vitro developmental study. J Biol Chem. 1991;266: Robbins J, Gulick J, Sanchez A, Howies P, Doetschman T. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. J Biol Chem. 1990;265:l Wang R, Clark R, Bautch VL. Embryonic stem cell-derived embryoid bodies form vascular channels: an in vitro model of blood vessel development. Development. 1992;114: Accili D, Taylor SI. Targeted inactivation of the insulin receptor gene in mouse 3T3-L1 fibroblasts via homologous recombination. Proc Natl Acad Sci USA. 1991;88: