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8 Animal Models: Genetic Manipulation

Karen M. Lyons

INTRODUCTION

Genetically manipulated mice have contributed enor-mously to our identifi cation of genes controlling skeletal development and to the clarifi cation of their mechanisms of action. Techniques are available to examine the effects of loss-of-function, gain-of-function, and altered struc-ture of gene products. The ability to introduce defi ned mutations has facilitated the production of animal models of human diseases, cell lineage studies, examina-tion of tissue-specifi c functions, and dissection of dis-tinct gene functions at specifi c stages of differentiation within a single cell lineage.

OVEREXPRESSION OF TARGET GENES

The fi rst widely used approach to study gene function in vivo was to produce transgenic mice that overexpress target genes. This requires the full-length coding sequence (cDNA) of a gene to be cloned downstream of a promoter. There are severa1 promoters that have been well charac-terized and used to drive gene expression in skeletal tissues. In the following sections, promoters that have been used to drive transgene expression directly are

described. The use of these and other promoters to enable overexpression or underexpression of target genes using Cre recombinase is discussed in a separate section.

Chondrocytes The most widely used cartilage-specifi c promoter is derived from the mouse pro aI(II) collagen gene ( Col2al ). This promoter drives high levels of expression beginning after the condensation stage in appendicular elements, and prior to condensation in axial elements in the sclero-tomal compartment [1] . The Col11a2 promoter has also been used to overexpress genes in chondrocytes, although some of these promoters also drive expression in peri-chondrium and osteoblasts [2, 3] . Overexpression in pre-chondrogenic limb mesenchyme has been achieved using the Prxl promoter [4] . Promoters that drive high levels of expression in hypertrophic chondrocytes have not been described. Chicken and mouse Col10a1 promoters allow transgene expression in hypertrophic chondrocytes at low to moderate levels; however, expression is not seen in all hypertrophic chondrocytes and is weak [5] . The recent development of Col10a1-Cre knock-in mice, when used in conjunction with Cre-inducible transgenic lines (see below) [6] may permit high levels of gene expression in these cells.

Introduction 69 Overexpression of Target Genes 69 Gene Targeting 70 Tissue-Specifi c and Inducible Knockout and Overexpression 71

Lineage Tracing and Activity Reporters 73 Functional Genomics 73 General Considerations 73 Acknowledgments 73 References 73

Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Eighth Edition. Edited by Clifford J. Rosen.© 2013 American Society for Bone and Mineral Research. Published 2013 by John Wiley & Sons, Inc.

70 Molecular, Cellular, and Genetic Determinants of Bone Structure and Formation

tissue-specifi c promoters allow easy visualization of spe-cifi c cell types in vivo and may permit their isolation with a resolution not possible using other methods. Other methods for detecting specifi c lineages based on site-specifi c recombination systems are discussed in a separate section.

A major caveat of the transgenic approach is that con-stitutive overexpression models often yield nonphysio-logical levels of protein expression, and this will confound interpretation of the role of the normal gene. Modifi ed transgenic approaches can overcome some of this uncer-tainty. These include the use of transgenes encoding dominant negative variants or natural antagonists. These approaches lead to loss of function and thus target path-ways in their normal physiological context.

The site of transgene integration can have major con-sequences on tissue specifi city and levels of expression. This can be exploited to examine dose-dependent effects, but care must be taken to assess not only levels of expres-sion but also sites of transgene expression, making com-parisons of multiple transgenic lines potentially diffi cult to interpret.

Finally, overexpression of genes that have profound effects in skeletal tissues may confer embryonic lethal-ity, precluding the establishment of stable transgenic lines. By the same token, transgenic lines that can be established overexpress genes only to an extent compat-ible with survival to sexual maturity. Several bigenic systems address this issue to permit establishment of lines that overexpress genes at levels that confer lethality. One uses the tetracycline (tet) responsive trans-activator [15] . This system permits tissue-specifi c tet-responsive gene expression. A transactivator (tTA) whose activity is modifi ed by tet or the tet analog doxycline (dox) is expressed under the control of a tissue-specifi c or ubiquitous promoter. The second component is a strain expressing the gene of interest under the control of the operator sequences of the tet operon (tetO). Depending on whether the transactivator is induced (tTA: tet-on) or repressed (rtTA: tet-off) by dox, expression of target genes can be induced or repressed. A second bigenic strategy uses the GAL4/UAS system. This uses one transgenic strain expressing the GAL4 transactivator under a tissue-specifi c or inducible promoter and a second transgenic strain expressing the gene of interest under the control of the UAS sequence, which requires GAL4 binding for activity. These systems have been used to develop trans-genic lines that permit activation of genes in cartilage and bone [16, 17] .

GENE TARGETING

The most widely used technique for genetic manipula-tion is gene targeting in mouse embryonic stem (ES) cells. Briefl y, a targeting construct contains a portion of the gene of interest, along with a modifi cation that renders the gene product inactive or modifi es its activity.

Osteoblasts Several promoters allow overexpression of genes in osteo-blasts. The most frequently used is a 2.3-kb fragment from the rat or mouse Col1a1 proximal promoter ( 2.3Col1a1 ). Strong and specifi c activity is seen in fetal and adult mature osteoblasts and osteocytes [7] . A second 2 .3Col1a1 promoter has been described [8] ; this promoter shows similar activity in bone, but expression in brain has recently been noted [9] . The 3.6-kb proximal Col1a1 promoter drives strong gene expression at an earlier stage of differentiation (preosteoblasts), but it is also expressed in nonosseous tissues, including tendon, skin, muscle, and brain [8, 9] . The 1.7-kb mouse Osteocalcin (OC; Bglap) promoter has been used to express genes in mature osteoblasts. However, this promoter is expressed in a low percentage of osteoblasts and at a relatively low level. Consistent with this, the 1.7-kb OC promoter is unable to drive Cre recombinase expression at effective levels [7] . On the other hand, 3.5- to 3.9-kb human OC pro-moter fragments drive osteoblast-specifi c expression in a large proportion of mature osteoblasts and osteocytes [10] . Osteocyte-specifi c promoters have not been widely used, although osteocyte-specifi c transgene everexpres-sion can potentially be achieved using Dmpl -Cre mice (see below) [11] .

Tendon and l igament Tendon patterning and differentiation are seldom studied genetically because of the lack of tissue-specifi c markers. Scleraxis ( Scx ) encodes a transcription factor expressed in developing tendons and ligaments and their progeni-tors. The development of Scx-Cre mice [12] provides a potential strategy for inducing expression of Cre-inducible transgenes (see below).

Osteoclasts There are a variety of promoters that drive high levels of gene expression in osteoclasts and their progenitors. These include CDllb , expressed in monocytes, macro-phages, and along the osteoclast differentiation pathway from monoculeated progenitor cells and into mature osteoclasts [13] , and TRACP , expressed in mature osteo-clasts and their precursors [14] .

Advantages and d isadvantages of o verexpression a pproaches The major advantages of the transgenic approach are that it is straightforward and inexpensive, high levels of gene expression can potentially be achieved, and transgenic mice often show obvious phenotypes. Furthermore, transgenic strains in which marker genes such as LacZ , GFP , and/or ALP are expressed under the control of

Animal Models: Genetic Manipulation 71

This is discussed below in the context of tissue-specifi c knockouts.

TISSUE-SPECIFIC AND INDUCIBLE KNOCKOUT AND OVEREXPRESSION

The ability to achieve site-specifi c recombination has revolutionized analysis of gene action in skeletal cells. Tissue-specifi c recombination circumvents the early lethality associated with global knockout or overexpres-sion. Tools are available that allow researchers to ablate or express genes in specifi c types of skeletal cells at spe-cifi c stages of commitment and differentiation.

Several methods can be used to achieve tissue-specifi c gene knockout or activation; these rely on site-specifi c recombinases derived from bacteriophage (Cre) or yeast (Flp) [20] . Cre and Flp recombine DNA at specifi c target sites. Depending on the orientation of the sites, the recombinase catalyzes excision or inversion of DNA fl anked by the sites. Two mouse lines are required. For the Cre- loxP system, these are the “fl oxed” strain, in which the region of the gene targeted for deletion is fl anked by loxP sites, and a second transgenic mouse line in which Cre recombinase is expressed under the control of an inducible and/or tissue-specifi c promoter. In mice carrying both the fl oxed gene and the Cre transgene, Cre deletes the sequence fl anked by loxP sites. The loxP sites are usually placed in introns and generally do not inter-fere with the normal function of the gene. Hence, the fl oxed target gene usually functions normally except in tissues where Cre is expressed.

This technology is most commonly used to inactivate genes in which a critical exon is fl anked by loxP sites. However, it has also been used to achieve site-specifi c inducible overexpression. In this case, a transgene is often generated under the control of a strong ubiquitious promoter such as CAG [21] . The expression of the trans-gene is prevented by placing a strong transcriptional stop signal, fl anked by loxP sites. The gene is activated when Cre catalyzes excision of the stop signal [22] . Engin et al. [23] provide an example of this approach in osteoblasts.

Most studies have used constitutively active forms of Cre. However, ligand-regulated forms enable temporal control of gene activity. The most popular strategy uses fusions of Cre to a ligand binding domain from a mutant estrogen receptor (ER) [24] . The ER domain recognizes the synthetic estrogen antagonist 4-OH tamoxifen (T), but is insensitive to endogenous β -estradiol. In the absence of T, the Cre-ER(T) fusion protein is retained in the cytoplasm. Binding of T to the ER domain induces a conformational change that permits the fusion protein to enter the nucleus and catalyze recombination. Many Cre strains have been developed that are suitable for analysis of skeletal tissues. A complete list of published Cre lines can be found on the Mouse Genome Informatics site (www.informatics.jax.org/recombinases). A few of the

Gene targeting in ES cells can also be used to generate knock-in models. In these, a locus of interest is modifi ed such that it encodes a gene product with altered activity. A major application of this technology has been to gener-ate mouse models of human genetic diseases.

The ability to eliminate or alter the structure of defi ned genes is possible in the mouse because of the unique properties of ES cells. These can be genetically manipu-lated in culture yet retain the ability to colonize the germline when injected into a mouse blastocyst. Once incorporated, they can give rise to germ cells, permitting the establishment of mouse strains carrying the modifi ed gene. ES cells from the 129 strain were the fi rst to be derived and are the most frequently used. However, 129 mice are poor breeders and exhibit abnormal immuno-logical characteristics [18] . Germline competent ES cell lines from 129, C57Bl/6, and C3H are available commer-cially from multiple sources. These strains have different bone mineral density (BMD) profi les [19] that must be taken into consideration when interpreting skeletal phenotypes.

Germline-competent ES cells carrying defi ned muta-tions in many genes are now readily available. These can be found by searching the Mouse Genome Informatics website (www.informatics.jax.org). The most time-consuming step in gene targeting is the introduction of modifi ed ES cells into the germline. This is most com-monly done by blastocyst injection, resulting in an F 0 animal that is partially derived from the modifi ed ES cells. These chimeric mice are bred to obtain F 1 mice that are heterozygous for the defi ned mutation. There are many university core facilities and commercial groups that routinely perform blastocyst injections.

Advantages and d isadvantages of g ene t argeting Phenotypes caused by loss of function provide direct insight into the physiological roles of the ablated gene product. Moreover, novel actions of targeted genes can emerge because, unlike transgenic models, global knock-out models are not limited to a particular tissue or system.

A complication of the global knockout approach is that the deletion of genes essential for early develop-ment may preclude analysis of the roles of these genes in skeletal tissues. On the other hand, many knockout strains do not exhibit obvious phenotypes because of functional redundancy; the creation of double or even triple knockouts may be necessary. Another consider-ation is that global knockout strains usually contain a modifi ed allele in which the selectable cassette used to screen the ES colonies is retained in the locus of interest. On occasion, this leads to effects on neighboring genes. These effects can be shown by comparing phenotypes of mice carrying null alleles in which the selection cassette is left in place with those in which it has been removed.

72 Molecular, Cellular, and Genetic Determinants of Bone Structure and Formation

discussed above, also targets tendon and fi brous cells types in the suture, skin, and several organs [8, 9] . Col1a1-Cre lines (2.3 kb) show more restricted expression to mature osteoblasts, but ectopic expression in the brain is noted in some lines [9] . Osteocalcin-Cre drives exci-sion in mature osteoblasts but is not activated until just before birth [10] . Few inducible Cre transgenic strains have been developed for bone. An inducible 2.3 Col1a1-ERT strain has been described [33] .

Osteoclasts Several Cre strains permit ablation in myeloid cells. These include LysMcre mice, in which Cre has been introduced into the M Lysozyme locus [34] , and a strain in which the CDllh promoter drives Cre expression in macrophages and osteoclasts [35] . Strains permitting Cre-mediated recombination in mature osteoclasts include a Cre knock-in into the Cathepsin K (Ctsk) locus [35] and transgenic lines expressing Cre under the control of the TRAPC and Ctsk promoters [36] .

Considerations w hen u sing i nducible k nockouts The most signifi cant advantage of the Cre- loxP system is its fl exibility, permitting exploration of gene function in multiple tissues at multiple time points. However, there are some caveats. It may be diffi cult to fi nd a pro-moter that drives Cre expression with suffi cient activity to result in complete excision of the target gene; this is highly dependent on the fl oxed allele. Moreover, Cre transgenic strains based on identical promoters but gen-erated in different laboratories can exhibit different spec-ifi cities and effi ciencies. For this reason, every study should include controls to verify the extent of Cre-mediated recombination of the fl oxed line of interest. Floxed genes also vary with respect to the kinetics of Cre-mediated recombination. This must be borne in mind when attempting to compare phenotypes caused by excision of different genes using the same Cre trans-genic line.

The presence of the drug selection cassette in a fl oxed gene can have a major effect on expression levels of the targeted allele even in the absence of the Cre transgene. Prominent examples include a fl oxed Fgf8 strain in which retention of the neo cassette leads to a hypomorphic (reduced function) phenotype [37] , and Scleraxis knock-outs, in which the presence of the drug selection cassette led to embryonic lethality by day 9.5 of gestation [38] . In striking contrast, mice homozygous for a Scleraxis null allele in which the drug selection cassette was removed are viable as adults [39] . Another important consider-ation is that in some cases, Cre itself may confer a phe-notype due to toxicity. This is especially true if the Cre is expressed as a fusion with GFP [40] .

With respect to inducible Cre models, the inducer may have a signifi cant impact on the phenotype. Both doxy-

most widely used, and some promising newcomers, are discussed below.

Uncondensed m esenchyme, m esenchymal c ondensations, and n eural c rest Prxl-Cre drives expression in early uncondensed limb and head mesenchyme [4] . Dermo1-Cre expresses Cre in mesenchymal condensations [25] . Sox9 is also expressed in mesenchymal condensations. A Sox9-Cre knock-in strain drives Cre-mediated excision in precursors of osteoblasts and chondrocytes in these condensations [26] . The Wnt1-Cre transgene drives expression in the migrating neural crest and can be used to ablate genes in all chondrocytes and osteoblasts derived from this source [27] .

Cartilage The most widely used Cre strain for cartilage is Col2a1-Cre [1] . The activity of this promoter seems to be restricted to chondrocytes in the majority of studies, but there appears to be a brief window during chondrogenesis when Col2a1-Cre is expressed in the perichondrium [28] . Hence, controls should be performed to determine the extent to which Cre is expressed in the perichondrium in specifi c experiments. To date, no published Cre lines exhibit suffi cient robustness to permit gene deletion in hypertrophic chondrocytes, but several groups are working on this.

The development of tools that permit inducible recom-bination in postnatal cartilage has been a challenge. Several groups have generated transgenic lines using CreER(T) fusion proteins under the control of the Col2al promoter. These strains permit ablation in articular chondrocytes if T is administered within 2 weeks after birth [28–30] . However, expression of Col2a1 is known to be low in adults, and although not all of these lines have been tested for in adults, in the one study examin-ing this, the effi ciency of recombination was very low [28] . A major breakthrough has been the development of Aggrecan-CreERT2 mice, in which CreERT2 has been knocked into the Aggrecan (Acan) locus [31] . These mice exhibit robust Cre expression in the adult growth plate and articular cartilage, as well as in fi brocartilage.

Osteoblasts The transcription factor Osterix ( Osxl ) is expressed in osteoblast precursors. A Cre-GFP fusion protein has been inserted into the Osxl locus on a BAC transgene [32] . This construct allows the targeting of osteoblast precur-sors. Several transgenic lines in which Cre is expressed under the control of the Col1a1 promoter permit exci-sion of genes in osteoblasts. A 3.6-kb Col1a1 promoter drives high levels of Cre expression in osteoblasts but, as

Animal Models: Genetic Manipulation 73

lished in 2007 to coordinate international efforts to generate conditional alleles for all mouse genes. As of 2011, ES cells carrying conditional alleles in nearly 8,000 genes have been generated. Mutant Mouse Regional Resource Centers (http://www.mmrrc.org) have permit-ted the acquisition and storage of another 320 strains developed in individual laboratories. A second major effort is the use of gene trapping, a high-throughput mutagenesis strategy. Progress on these and other muta-genesis efforts can be found on the Mouse Genome Infor-matics and IMKC websites [49, 50] .

GENERAL CONSIDERATIONS

With all of the successes in genetic manipulation, the real bottleneck is phenotyping. Phenotype is dependent on genetic and environmental factors. As discussed, inbred strains vary considerably in their peak BMD. Moreover, housing conditions and food intake can have a signifi cant impact on metabolic parameters and BMD [51, 52] . Hence, even genetically identical mice can have different phenotypes in different facilities. It is important to assess phenotypes at different ages because effects present at early stages may be compensated for later on. An example of this can be seen in matrix metalloprotein-ase (MMP)-9-defi cient mice [53] . These exhibit prenatal expansion of the hypertrophic zone but are normal within a few weeks of birth. In contrast, other phenotypes mani-fest only in late stages or when a metabolic stress is applied.

Caution must be taken when extrapolating fi ndings in mouse models to functions in humans. Biomechanical loading and hormonal effects on bones are clearly differ-ent in mice and humans. Moreover, linear growth in humans ceases after epiphyseal closure, whereas in mice the growth plate does not fuse. Nonetheless, similarities outweigh the differences by far, and genetic models are likely to play an increasingly prominent role in every aspect of research in skeletal biology.

ACKNOWLEDGMENTS

The author acknowledges funding from NIH (AR052686 and AR044528).

REFERENCES

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cycline and tamoxifen can have profound effects on car-tilage, bone, and osteoclasts independently of target gene deletion. Even the low doses of tamoxifen used to cata-lyze Cre-ER(T)-mediated excision may have effects on bone [41] . Thus, a control group of Cre-negative mice treated with the inducer may need to be included to examine the impact of this variable on the mutant phe-notype under study.

LINEAGE TRACING AND ACTIVITY REPORTERS

Genetically modifi ed mice have permitted the determi-nation of cell lineage relationships and the relative con-tributions of cells from various sources to a given organ with unprecedented resolution. These studies rely on strains that carry a fl oxed reporter gene, such as LacZ or GFP . For example, the R26R strain carries a fl oxed LacZ cassette introduced into the ROSA26 locus [42] . When bred to a Cre-expressing strain, all cells in which Cre is expressed, and all of their descendants, express LacZ. R26R mice have been used to test the specifi city and effi ciency of Cre-expressing transgenic lines, although use of this strain in bone is limited by the fact that osteo-blasts express endogenous LacZ.

Major insights have been made using R26R to study osteoprogenitors. For example, Wntl-Cre;R26R mice revealed that frontal bones are derived from the neural crest, but parietal bones are derived from mesoderm [43] . A second example is the demonstration that immature osteoblasts move into developing bone along with invad-ing blood vessels [44] . Most recently, lines have been generated that permit live imaging of the expression of fl uorescent proteins in specifi c organelles (cell mem-brane, nucleus, etc.) in a tissue-specifi c and inducible manner; this system was used to mark sites of Sox9-Cre expression in skeletal tissues [45] .

Transgenic reporter lines can also be used to monitor signaling pathway activity. For example, Wnt pathway activity has been monitored in vivo using TOPGAL mice to track β -catenin activity during endochondral bone for-mation [46] . Tools are also available to monitor canonical bone morphogenetic protein (BMP) pathway activity in vivo [47, 48] .

FUNCTIONAL GENOMICS

Functional data are available for nearly 14,000 of the estimated 25,000 mouse genes. This information can be accessed through the Phenotype/Alleles project in Mouse Genome Informatics (www.informatics.jax.org/phenotypes). However, fewer than 4,000 of the annota-tions relate to gene function in skeletal tissue. Thus, there is much work remaining to be done. The Interna-tional Mouse Knockout Consortium (IMKC) was estab-

74 Molecular, Cellular, and Genetic Determinants of Bone Structure and Formation

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