Selective Alterations in Organ Sizes in Mice with a Targeted Disruption of the Insulin-Like Growth Factor Binding Protein-2 Gene

Teresa L. Wood1, Leslie E. Rogler2, Maureen E. Czick, Alwin G.P. Schuller and John E. Pintar

Department of Neuroscience and Cell Biology Robert Wood Johnson Medical School University of Medicine and Dentistry New Jersey Piscataway, New Jersey 08854


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-like growth factor binding protein 2 (IGFBP-2) is one member of the family of IGF binding proteins believed to have both endocrine functions elicited by modulating serum IGF half-life and transport as well as autocrine/paracrine functions that result from blocking or enhancing the availability of IGFs to bind cell surface receptors. To clarify the in vivo role of IGFBP-2, we have used gene targeting to introduce a null IGFBP-2 allele into the mouse genome. Animals homozygous for the altered allele are viable and fertile, contain no IGFBP-2 mRNA, and have no detectable IGFBP-2 in the adult circulation. Heterozygous and homozygous animals showed no significant differences in prenatal or postnatal body growth. Analyses of organ weights in adult males, however, revealed that spleen weight was reduced and liver weight was increased in the absence of IGFBP-2. In addition, ligand blot analyses of sera from adult IGFBP-2 null males showed that IGFBP-1, IGFBP-3, and IGFBP-4 levels were increased relative to wild-type mice. These results demonstrate that up-regulation of multiple IGFBPs accompanies the absence of IGFBP-2 and that IGFBP-2 has a critical role, either directly or indirectly, in modulating spleen and liver size.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of growth factor and hormone action by soluble binding proteins has emerged as a common theme for a wide variety of peptides including insulin-like growth factor (IGF)-I and -II, transforming growth factor-ß (TGF-ß), activin, epidermal growth factor (EGF), nerve growth factor (NGF), tumor necrosis factor (TNF), GH, and CRF (1, 2, 3, 4, 5, 6, 7). Among the best studied of these binding proteins are the IGF binding proteins (IGFBPs). Six different, high-affinity IGFBPs have been identified that each derive from a unique member of the IGFBP gene family (5, 8, 9). Most IGFBPs are found at high levels in adult circulation where they regulate IGF half-life and availability (10, 11, 12). In addition, in vitro studies suggest that the IGFBPs may have autocrine/paracrine actions by localizing IGFs to specific tissue sites and by modulating IGF binding to cell surface receptors or to the extracellular matrix (ECM) (5, 9, 13, 14, 15). Although some members of the IGFBP family are coexpressed in specific cell types, each of the six members of the IGFBP family has a distinct expression pattern in both developing and adult tissues (16, 17, 18, 19, 20, 21, 22, 23). Moreover, the major IGFBPs in adult serum are regulated differentially (5, 9, 12, 24, 25, 26, 27). These data support the hypothesis that each member of the IGFBP family has a unique function.

IGFBP-2 is a particularly prominent IGFBP in late fetal and neonatal serum (28, 29, 30). We have previously reported high IGFBP-2 expression in specific embryonic tissues and cell types (19, 31, 32, 33). IGFBP-2 mRNA is highly expressed in the epiblast of the egg cylinder and its derivatives including neuroectoderm, surface ectoderm, and the sensory placodes. Expression of IGFBP-2 also is found in specific endodermal derivatives including the developing gut and liver. In contrast, fetal mesoderm derivatives lack IGFBP-2 expression with the striking exceptions of the notochord, mesonephric tubules, and a region of modified mesoderm, termed the anterior splanchnic mesodermal plate (ASMP), that is the progenitor of the spleen (32). In the adult, IGFBP-2 is found at significant levels in serum where it is under complex physiological regulation (5, 9, 12, 14, 24, 25, 26). For example, liver IGFBP-2 mRNA expression and circulating levels of IGFBP-2 are increased by IGF-I (34), by hypophysectomy (35), and by conditions that cause mild insulin deficiency (24). IGFBP-2 is also expressed in the adult central nervous system (CNS) in the choroid plexus and neural lobe of the pituitary (19), and IGFBP-2 protein is a component of cerebral spinal fluid (36).

Despite a large amount of in vitro data, the precise in vivo function(s) of IGFBP-2 as well as the other IGFBPs has been difficult to address. The utilization of homologous recombination in embryonic stem (ES) cells to create null mutations in mice has resulted in valuable information on the in vivo function of many molecules including IGF-I, IGF-II, and their receptors (37, 38, 39, 40, 41, 42, 43, 44, 45). We here report the production and characterization of a mouse line that carries a null mutation in the IGFBP-2 gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted Disruption of the Mouse IGFBP-2 Gene
A {lambda} clone containing exons 2, 3, and 4 of the mouse IGFBP-2 gene was isolated from a C57Bl6/J genomic library (Fig. 1AGo). To generate a null mutation of the BP-2 gene by homologous recombination, we designed the targeting vector to delete essentially all of exon 3 (115 nucleotides, representing 38 amino acids) as well as 0.6 kb of the 3'-flanking intron including the exon/intron boundary (Fig. 1BGo). Addition of the neomycin resistance gene introduced two novel, diagnostic EcoRI restriction sites (Fig. 1BGo). The targeting vector was linearized at a unique nonplasmid BamHI site and used to electroporate ES cells. Seven prospective ES clones were identified initially by PCR screening (Fig. 1DGo) and subsequently were confirmed to have targeted BP-2 alleles by Southern blot analysis. Six of the seven confirmed recombinant clones contained a normal chromosome complement while one clone was triploid. The six clones with normal karyotypes were tested for their ability to contribute to chimeric mice by microinjection into C57Bl6/J blastocysts and subsequent transfer of injected blastocysts into pseudopregnant females. Two ES lines successfully contributed to chimeric males that transmitted the mutant allele through the germ line. Progeny heterozygous for the mutated gene, identified by Southern analysis of tail DNAs, were mated, and offspring from these heterozygous matings were genotyped as neonates. All genotypes were represented in the progeny from these matings (Fig. 1EGo) in proportions not significantly different from Mendelian expectations (Table 1Go). We have not detected any differences in viability, fecundity, or longevity in mutant homozygote animals when compared with wild-type littermates over more than 10 generations.



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Figure 1. Targeted Deletion of the IGFBP-2 Gene

Panel A, Restriction map of the wild-type mouse IGFBP-2 genomic fragment containing exons 2, 3, and 4. Restriction sites are: B, BamHI; D, DraI; E, EcoRI; E47, Eco47II-I; H, HindIII; P, PstI;.T, Tth 111I. Panel B, Replacement vector for deletion of the IGFBP-2 gene. The neomycin resistance (NeoR) gene was used to replace a DNA fragment that contained the majority of BP-2 exon 3 (115 nucleotides, representing 38 amino acids) as well as 0.6 kb of the 3'-flanking intron including the exon/intron boundary. The insertion of the NeoR gene resulted in the addition of two novel, diagnostic EcoRI restriction sites. The targeting vector was linearized at a unique nonplasmid BamHI site and used to electroporate ES cells. Panel C, Predicted structure of mutated IGFBP-2 locus upon homologous recombination. The dashed line represents the 2.3-kb PCR fragment amplified using an upstream primer to BP-2 gene sequence outside the targeting vector and a downstream primer in the neoR gene. Panel D, Ethidium bromide-stained gel showing PCR analysis of ES clones after electroporation and selection in G418 and gancyclovir. Lanes 1–7 show the results of PCR amplification of seven different pools of ES lines. Each pool represents DNAs from five ES clones. Lanes 2 and 7 show the 2.3-kb amplified product expected after homologous recombination. Subsequent PCR analysis of individual ES clone DNAs from each positive pool identified two positive ES cell clones that were confirmed as homologous recombinants at the BP-2 locus by Southern hybridization. Panel E, Southern blot of EcoRI-digested tail DNAs showing that three genotypes arise from heterozygote crosses. Blot was hybridized with a random-primed 32P-labeled cDNA probe containing exons 2, 3, and 4 of the IGFBP-2 gene. +/+, Single 7-kb EcoRI fragment; EcoRI fragment denotes normal allele; +/-, 7-kb normal allele and two fragments (4.8 kb, 2.8 kb) that correspond to the mutant allele; -/-, only the 4.8-kb and 2.8- kb fragments are detected from the mutant alleles.

 

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Table 1. Transmittance of the Mutant BP2 Allele

 
BP-2 mRNA and Protein Are Absent in the Homozygous BP-2 Mutant Animals
Tissues from adult animals of all three genotypes were analyzed for IGFBP-2 mRNA and protein by Northern and Western blot analyses (Fig. 2AGo, B). Mice heterozygous for the mutated allele contained reduced levels of BP-2 mRNA in brain and liver and of IGFBP-2 protein in serum. The reduction in IGFBP-2 protein and mRNA occurred whether the mutant gene was maternal or paternal in origin suggesting that, unlike IGF-II (37) and the IGF-II/mannose-6-phosphate receptor (46), the IGFBP-2 gene is not imprinted. Homozygous mutants contained no detectable BP-2 mRNA or immunoreactive protein of either native size or altered size, which conceivably could have been produced by alternate splicing around the neomycin gene (47).



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Figure 2. IGFBP-2 RNA and Protein Levels in Heterozygote and Homozygote Mutants

A, Northern blot of 20 µg total RNA isolated from brain or liver of adult wild-type (+/+), heterozygous (+/-), or homozygous (-/-) IGFBP-2 mutant mice. Blot was hybridized with a random-primed 32P-labeled probe to rat IGFBP-2 and exposed to X-OMAT AR film. The probe detected a single band at approximately 1.9 kb. Level of RNA in each lane was verified by reprobing the blot with a random-primed labeled cDNA to mouse actin which hybridized to a single RNA species at approximately 2.1 kb. B, Western blot of adult serum proteins separated by PAGE and incubated with a rabbit antibody to rat IGFBP-2 (1:1000) followed by an alkaline phosphatase-conjugated antirabbit secondary antibody as described in Materials and Methods. The immunopositive band is approximately 30 kDa. Shown are representative serum samples from two wild-type(+/+), one heterozygote (+/-), and four homozygote (-/-) mutant mice.

 
Developmental Expression of Other IGFBPs
In several previous gene disruption experiments, ablation of one member of a gene family has resulted in compensation by one or more other members of the gene family. Functional redundancy has been demonstrated for several gene families including the muscle transcription factors Myo D, Myf-5 and myogenin (48, 49, 50, 51), the retinoic acid receptors (52, 53, 54), the src family of tyrosine kinases (55), and the MAD family of transcription factors (56). In some cases, compensation by another family member has been accompanied by an increase in expression of the corresponding gene (48, 57). Of potential significance for interpretation of the BP-2 mutant, previous studies have shown substantial overlap between the patterns of BP-2 and BP-5 expression during prenatal development (19, 23). To determine whether any changes in BP-5 levels accompanied the BP-2 mutation during this period, levels of BP-5 mRNA in heads and trunks of embryonic day 13.5 and 17 BP-2 heterozygote and homozygote littermates were determined by solution hybridization (Fig. 3Go). Each RNA sample was hybridized concurrently with both 32P-labeled BP-5 and actin antisense riboprobes to ensure accurate normalization (see Materials and Methods). No significant differences were observed in levels of IGFBP-5 RNA in homozygote vs. heterozygote embryos at either embryonic age. However, since RNA was isolated from large fetal regions, it is possible that regional or cellular changes in BP-5 RNA levels could have been missed in these experiments. Since previous studies also demonstrated specific expression patterns for other IGFBP genes during embryonic development (16, 17, 23), we extended the analysis of BP-2 minus embryos to determine whether tissue-specific expression patterns of BP-5 as well as other BPs were altered in the absence of BP-2. In situ hybridization was used to analyze expression patterns of the other IGFBPs and of IGF-II in BP-2 homozygous mutant embryos during midgestation. To date, we have observed no ectopic expression or gross change in the spatial pattern or level of mRNAs for any IGFBPs or for IGF-II in e15 embryos (data not shown).



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Figure 3. Analysis of IGFBP-5 RNA Levels in BP-2 Homozygote and Heterozygote Embryos

The relative levels of IGFBP-5 mRNA in heads (H) or trunks (T) of heterozygous and homozygous littermates are shown at embryonic days 13.5 and 17. Total RNA was isolated from heads and trunks of homozygote and heterozygote littermates. Each embryo was genotyped by Southern analysis of placental DNA. Individual RNA samples (5 µg from each head or trunk) were hybridized with both 32P-labeled mouse BP-5 and mouse ß-actin riboprobes. Resulting protected bands were excised from the dried gels and counts per min were determined from scintillation counting. BP-5 measurements were corrected based on the actin mRNA levels to normalize for variation in RNA amounts between samples. Graph shows mean ± SE for each group. Three to five animals were analyzed per group. Student’s t test was performed to determine significance between heterozygote and homozygote embryos for each region at each age. No significant differences were observed in levels of BP-5 RNA in homozygote vs. heterozygote embryos at these ages.

 
Body Growth in IGFBP-2 Null Mutants
Gene targeting of IGF-I, IGF-II and the type I IGF receptor have all resulted in prenatal growth deficits (37, 38, 39, 40, 43). IGF-I deficiency results in additional defects in postnatal growth (38, 39, 40, 43). To determine whether the absence of IGFBP-2 resulted in altered prenatal growth, mice from multiple heterozygote crosses were weighed at birth (Fig. 4Go). There was no difference in birth weights among the three genotypes, suggesting that prenatal growth was not affected by the mutation. Mice of all genotypes were then weighed daily until postnatal day 14 (p14) and then every 2–3 days through p35. Body weights of both male (Fig. 4AGo) and female (Fig. 4BGo) wild-type and homozygous mutant mice, as well as heterozygous mice (data not shown), were indistinguishable during this period, demonstrating that the extent of postnatal growth was also unaffected by the BP-2 mutation.



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Figure 4. Postnatal Growth Curves of Wild-Type and BP-2 Homozygous Mutant Mice

Male (A) and female (B) body weights of mice from heterozygous matings. Mice derived from heterozygote matings were weighed daily beginning on the day of birth (p0) through day 14 and then every second or third day until day 35.

 
Organ Weights in IGFBP-2 Homozygous Null Mutants
Analysis of transgenic mice that overexpress IGF-I or IGF-II has shown that these peptides induce growth of specific organs (58, 59, 60, 61, 62). To determine whether absence of IGFBP-2 caused selective changes in organ growth, numerous organs from mutant and wild-type adult males were weighed (Table 2Go). Specific organs analyzed included liver, kidney, heart, lung, and spleen. Since previous studies have shown that weight of some, but not all, organs correlate with changes in body weight (59, 60, 61), organ weights from the two groups were analyzed both directly or as a function of body weight. Using either analysis, spleen weights of IGFBP-2 homozygous mutants were significantly less than that of wild-type animals (P <= 0.001; Table 2Go). Weights of kidneys and hearts were significantly reduced only when analyzed as a function of body weight (P < 0.001; Table 2Go). In contrast, liver weights in the BP-2 mutant animals were significantly higher than those of the wild-type animals both directly (P < 0.001) and as a function of body weight (P <= 0.005; Table 2Go).


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Table 2. Body and Organ Weights of Normal and BP2 Null Mutant Mice

 
Since the phenotype of several knock-out lines of mice have differed depending on the strain background (38, 63, 64), we have continued backcrossing the BP-2 mutation into the C57BL/6J mouse strain. Analysis of BP-2 minus and wild-type mice after seven generations of backcrossing revealed that BP-2 minus spleens were approximately 80% normal size (n = 4 +/+; 5 -/-), similar to the reduction seen previously in the outbred line of mice.

Analysis of Spleen Deficit in BP-2 Null Mutant Mice
The spleen arises developmentally from the ASMP, a region of the splanchnic mesoderm (65). Early studies of the mouse mutant Dominant Hemimelia demonstrated that a defect in the ASMP resulted in loss of the spleen (65) and, recently, it was determined that expression of the homeobox gene, Hox11, in the ASMP is essential for spleen formation (66, 67). Our previous studies showed expression of BP-2 in the ASMP during the embryonic period critical for spleen formation (32). To determine whether loss of BP-2 resulted in a deficit in early spleen growth, we weighed spleens from normal and BP-2 minus neonatal animals at postnatal day 12. The weight of BP-2 mutant spleens (31.7 ± 1.7 mg; n = 7) was not significantly different from those of normal mice (30.8 ± 0.8 mg; n = 6) at this age.

To begin to address whether the reduced weight of the adult BP-2 null spleens resulted in functional deficits, spleens from normal and BP-2 mutant mice were analyzed for histological alterations and for alterations in lymphocyte numbers. No gross histological abnormalities were observed in fixed sections from BP-2 mutant and wild-type spleens that were analyzed by light microscopy after hematoxylin and eosin staining. To assess whether the reduction in spleen size was correlated with a reduction in lymphocyte number in the BP-2 null mutant mice, spleens from four normal and four BP-2 homozygous mutant male mice were isolated, weighed, and homogenized to obtain lymphocytes (68). The total number of lymphocytes per spleen was reduced in the BP-2 homozygous mutants (7.5 x 107 ± 0.4) as compared with wild-type mice (1 x 108 ± 0.8; P = 0.05) in proportion to the observed reduction in spleen weights.

Serum Levels of Multiple IGFBPs Are Increased in IGFBP-2 Minus Mice
Circulating levels of IGFBPs in normal and BP-2 mutant animals were assessed using ligand blots. Serum was collected from age-matched (from 4–8 months) adult male normal (n = 11) and BP-2 homozygous mutant (n = 20) mice. Prominent serum bands that bound both [125I]-IGF-II and [125I]-IGF-I corresponded to predicted sizes for the mouse BP-3 doublet and mouse BP-4 (Fig. 5AGo). When the intensities of the radiolabeled bands were compared with Coomassie stained gels of identical samples, it was concluded that there was a 2.5-fold elevation of BP-3 and BP-4 levels in BP-2 mutant serum. Western immunoblots of the same serum samples confirmed the identity of the doublet as BP-3 and further demonstrated elevation of circulating BP-3 (Fig. 5BGo).



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Figure 5. Serum Levels of Other IGFBPs in Adult IGFBP-2 Homozygous Mutant Mice

A, Ligand blot showing adult serum IGFBPs after PAGE and incubation with 125I-IGF-II. Samples are identical to those seen in Fig. 2BGo. Prominent serum bands that bind to IGF-II correspond to predicted sizes for the IGFBP-3 doublet at 39–43 kDa (arrowhead) and to IGFBP-4 at 24 kDa (arrow). Based on comparisons with Coomassie-stained gels of identical samples, levels of both BP-3 and BP-4 are elevated in the homozygous BP-2 mutant mice. B. Western immunoblot to detect IGFBP-3 protein in serum samples shown in panel A. A second set of the serum samples shown in panel A were separated by PAGE and used for Western immunoblotting with a rabbit polyclonal antibody to rat IGFBP-3 (1:500) as described in Materials and Methods. The antibody detected a doublet at 39–43 kDa that was more intense in the BP-2 homozygote mutant serum compared with wild-type serum.

 
In separate experiments, sets of adult male normal and homozygote mutant animals (three to five animals, age 5–8 months) were assayed by ligand blotting using a gel system modified from that shown in Fig. 5Go (see Materials and Methods). Serum samples from three representative animals (two homozygote; one normal) revealed increased levels of the 38–45 kDa doublet (confirmed as BP-3) and a 24-kDa band identified by size as BP-4 (Fig. 6AGo). In addition, an enlarged image of the region that corresponds to 30–32 kDa shows BP-2 in the two wild-type samples and absent in the homozygous sample (confirmed by immunoblotting, data not shown). In addition, levels of two additional bands (arrows) of lower molecular mass than BP-2 appeared increased in the homozygous serum. The lower of these bands was identified as IGFBP-1 by immunoblotting (data not shown), while the additional radiolabeled band was increased in the region of the gel where a glycosylated form of BP-4 has been reported (69, 70). To determine whether the increases in serum levels of BP-3 and BP-4 were due to induction of the respective mRNAs for these proteins, mRNA expression levels of BP-3 and BP-4 were analyzed by Northern blotting. After adjustment for hybridization to a control RNA, no significant differences were observed in liver RNA expression levels for either BP-3 or BP-4 in the homozygote BP-2 mutant animals as compared with wild-type animals (data not shown).



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Figure 6. Multiple BPs In BP-2 Region Are Up-Regulated In Adult Mutant Serum

Two different sets of adult male wild-type and homozygote mutant animals (three to five animals of each genotype) were assayed by ligand blotting using an expanded electrophoresis procedure to optimize separation in the 25- to 40-kDa protein range (see Materials and Methods). Three representative samples (two homozygote; one wild-type) are shown after ligand blotting with 125I-IGF-I. The upper doublet at 39–43 kDa was confirmed by Western immunoblotting to be BP-3 and the lower band (at ~24 kDa) corresponds to the known molecular mass of BP-4. The increased gel resolution reveals several bands in the region from 29–33 kDa. A band corresponding to BP-2 is seen in the +/+ sample only but is absent from the homozygote mutant sera. In addition, two additional bands of lower molecular mass than BP-2 are detectable in the homozygote mutant serum samples. These bands correspond to BP-1 and phosphorylated BP-4.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the targeting of a null allele to the mouse IGFBP-2 locus and the production of mouse lines in which the capability to produce IGFBP-2 has been eliminated. These strains represent the first in which one member of the IGFBP family has been genetically inactivated. Homozygote mutant animals are viable and fertile and can be distinguished from wild-type mice histopathologically by a 25% reduction in spleen size and a 15% increase in liver size. Although IGFBP-2 is expressed in the progenitor of the spleen during midgestation development (32), comparison of spleen weights in BP-2 null mutants at neonatal ages suggests that the reduction in spleen size in the BP-2 null mutant adult animals is not due to a developmental growth defect. Since BP-2 is expressed in the adult spleen (71), the reduction in spleen size in the BP-2 (-/-) animals may be the result of local actions of BP-2 on pubertal spleen growth or on adult spleen function. Previous studies have shown that spleen size is acutely responsive to changes in circulating levels of IGF-I in transgenic mice that overexpress IGF-I (59, 61), mice selected on the basis of high or low circulating levels of IGF-I (72), and mice or rats given exogenous IGF-I (73, 74, 75, 76). In all cases, high circulating levels of IGF-I resulted in increased spleen weight. These data suggest that the reduction in spleen weight in the BP-2 null mutant mice may be the result of decreased availability of IGF-I in these mice. Initial analyses of IGF-I serum levels in the BP-2 null mutants showed no significant differences in total IGF-I levels from the wild-type mice (A. J. D’Ercole, personal communication). Moreover, we observed no substantial differences in body growth in the BP-2 minus animals, supporting the conclusion that the absence of BP-2 has not resulted in significant alterations in circulating IGF levels. This suggests that the absence of BP-2 may result either in decreased transport of circulating IGF-I to the spleen or in decreased autocrine/paracrine actions of IGF-I in the spleen itself.

If BP-2 is required in spleen function, we postulate that the absence of BP-2 will cause specific deficits in spleen immune responses. Previous reports have demonstrated an increase in the ratio of CD4+ to CD8+ T cells in both rats and monkeys after infusion of exogenous IGF-I (73, 77). We have determined that total lymphocyte numbers are reduced in the spleens of the BP-2 mutant mice as compared with control mice, and that the reduction is proportional to the observed decrease in spleen weight. Preliminary studies analyzing the proportion of lymphocyte subsets suggest that the CD4 to CD8 ratio may be increased in the BP-2 minus spleens. Additional preliminary analyses of the immune response to herpes virus infection suggest that the BP-2 minus spleens are able to respond to viral challenge as assessed by increased spleen weight and lymphocyte number; however, levels of cytokine production by the mutant spleen lymphocytes may be altered. Future experiments are designed to correlate deficits in spleen function with pathogenesis of viral infection in the BP-2 mutant mice. However, since ratios of lymphocyte populations and response to viral challenge differ significantly in different mouse strains, we have postponed further analyses of spleen function in the BP-2 null mutants until backcrossing the mutation 10 generations into the C57Bl6/J strain has been achieved.

In contrast to the reduction in spleen size in the BP-2 null mutant mice, the livers of these mice show a significant increase in wet weight. The induction in liver weight in the BP-2 null mutants may be due to local autocrine/paracrine actions of BP-2 in liver growth since BP-2 is expressed at high levels in liver from the onset of its development. The mechanisms by which the loss of BP-2 results in opposite effects on spleen and liver size are unclear. Since attainment of normal liver size is particularly dependent on GH (58, 60), one possibility would be that slightly elevated GH levels in the IGFBP-2 null mice, insufficient to alter body size, could produce selective overgrowth of the liver. In fact, GH levels in both urine and serum are not significantly different between wild-type and IGFBP-2 mutant genotypes, with a tendency toward lower levels in the mutants (T. L. Wood, A. G. P. Schuller, and J. E. Pintar, unpublished observations). It thus seems more likely that the differential phenotypes in spleen and liver reflect differential functions of BP-2 in the growth and/or function of each organ. Data from in vitro studies have supported a role for the IGFBPs both in blocking and enhancing actions of IGFs. The ability of the IGFBP to inhibit or enhance IGF action has been proposed to depend upon whether the binding protein is free in solution or is cell surface or ECM associated, respectively (5, 9, 13, 14, 15). IGFBP-2 has the potential to bind cell surface integrins through its carboxy-terminal RGD sequence (5, 9, 14). In addition, a recent study has shown that BP-2, once bound to IGF-I or IGF-II, can bind to heparin and ECM (78). Whether IGFBP-2 predominantly enhances or inhibits IGF actions in vivo is unknown; however, the data presented here suggest that its role may be distinct in different tissues.

Targeted mutations in IGF-I (38, 39, 40, 43), IGF-II (37), and the type I IGF receptor (38, 39) all result in fetal growth deficits. In contrast, deletion of the IGF-II/M6P receptor (41, 42) results in fetal overgrowth due to excess IGF-II. Since the IGFBPs are believed to modulate the half-life and cellular actions of the IGFs, and since IGFBP-2 is prominently expressed during embryonic development, we predicted that the absence of circulating BP-2 might result in alterations in fetal growth. This prediction was not supported by the observed phenotype of the BP-2 minus mice that had neonatal weights indistinguishable from wild-type littermates. In addition, the expression of BP-2 in numerous sites in the embryo predicts a role for this IGFBP in development of many tissues where no gross phenotype was observed in the BP-2 null mutant mice. Several implications of this observation can be mentioned. First, we and others have shown that all other IGFBPs are expressed throughout the midgestational period in the embryo proper and thus may be able to compensate for the lack of IGFBP-2 (16, 17, 18, 19, 20, 23). In addition, other IGFBPs are also potentially available to the developing fetus from the decidualizing regions of the uterus (21). For example, IGFBP-3 is found lining the endothelial cells of the decidua, while IGFBP-1 lines the uterine lumen as early as embryonic day 8 in the rat. Thus, compensation for IGFBP-2 absence would not necessarily require an increase in levels of other IGFBPs, although in the adult BP-2 knockout mice, we did observe induction of circulating levels of several other members of the IGFBP family including IGFBPs-1, -3, and -4.

If the induction of the other IGFBPs in the BP-2 deletion mutant represents functional compensation, then multiple knockouts will be required to demonstrate additional roles for the IGFBPs. The necessity of multiple knockouts to delineate the functions of gene products that arise from multiple gene families has been repeatedly demonstrated. For example, while mice containing an ablation of only one isoform of the retinoic acid receptor (RAR-{gamma}-2) are normal, ablation of all {gamma}-RAR isoforms results in lethality (52, 53). In addition, genetic disruption of individual members of the src family of tyrosine kinases (Src, Fyn, and Yes) each produces a mild phenotype but combinatorial deletions of any two of these genes results in a more severe phenotype (55). Finally, genetic ablation of MyoD, which itself can induce muscle differentiation in fibroblast lines after transfection, has no dramatic effect on skeletal muscle differentiation; neither does ablation of a second transcription factor, Myf-5 (79). However, a double mutant of MyoD and Myf-5 produces a complete absence of skeletal muscle (49). These examples of functional compensation between related gene products provide a potential framework for understanding the restricted alterations in the IGFBP-2 mice that have thus far been observed. We suspect that mice lacking IGFBP-2 have adapted physiologically to its absence likely as a result of the up-regulation of other members of the IGFBP gene family. It is possible, however, that specific physiological challenge may reveal additional phenotypic alterations even in the absence of multiple gene targeting events.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeting Vector and Generation of Mice with a Null Mutation in the IGFBP-2 Gene
A mouse genomic clone containing exons 2, 3, and 4 of the IGFBP-2 gene was isolated from a {lambda} library (FIX II vector, Promega Corp., Madison, WI) that contained 15- to 20-kb inserts of mouse 129/ReJ genomic DNA. Restriction mapping and partial sequencing (Sequenase-version 2.0) confirmed the identity of the genomic clone and provided information for constructing the BP-2 targeting vector. To construct the targeting vector, two fragments from the mouse genomic clone were subcloned into a pBS (sk)II cassette (obtained from Dr. Steven Potter, University of Cincinnati, Cincinnati, OH). The targeting vector contained a positive selection marker, the neomycin resistance (neoR) gene, and a negative selection marker, the herpes simplex virus thymidine kinase (HSV-TK) gene, each driven by the HSV-TK promoter. Initially, a 1.4-kb Dra/Tth 111I fragment was subcloned at the 3'-end of the neoR gene and, subsequently, a 4.7-kb Eco47II-I/EcoRI fragment was subcloned between the neoR gene and the TK gene. The DNA for electroporation was purified by cesium chloride density centrifugation, linearized at the BamHI site, phenol-chloroform extracted, and ethanol precipitated before resuspension in sterile TE.

Linearized DNA was electroporated into ES cells grown to 50–60% confluence on mitomycin C-treated STO fibroblast feeder cells. ES clones that survived double selection in G418 (220 µg/ml) and gancyclovir (2 µM) were expanded and analyzed for homologous recombination at the BP-2 locus by PCR and Southern analyses. Correctly targeted ES cell clones were used for injections into blastocysts isolated from C57BL/6J mice. Injected blastocysts were transferred into the uterine horns of pseudopregnant mothers. Three resulting chimeric male mice demonstrated germ line transmittance of the mutant BP-2 allele and were used to generate progeny for analysis. A line of mice from one of these chimeras was expanded by backcrossing to both C57BL/6J and 129/ReJ females to generate a mixed strain (129/C57) background line and an inbred (129) background line carrying the BP-2 mutation. Genotyping of all mice was by Southern blotting of tail DNA. All mice were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

PCR Amplification and Southern Analysis
DNA was isolated from ES cells by lysis at 37 C for 15–20 min in 0.5 ml 0.05 M Tris-HCl (pH 7.5), 0.05 M EDTA, 0.1 M NaCl, 5 mM dithiothreitol (DTT), 1% SDS, and 0.5 mM spermidine containing 400 µg/ml proteinase K followed by incubation overnight at 55 C. The DNA was extracted with phenol/chloroform and then ethanol precipitated. Tail DNAs were isolated by incubation overnight at 50 C in 50 mM Tris (pH 8.0), 100 mM EDTA, and 0.5% SDS containing 30 µg/ml proteinase K followed by extraction with phenol/chloroform and ethanol precipitated.

Aliquots of DNAs from 5–10 individual double-selected ES cell clones were pooled for PCR analysis. Pooled DNA was used for PCR amplification using an upstream primer designed to a region of intron A of the BP-2 gene that was 5' to sequences included in the targeting vector (5'-TGCACACAGTTGGCTCTATG-3') and a downstream primer designed to sequences in the neomycin resistance gene (5'-ACAGACAATCGGCTGCTCTG-3'). Amplification was for 35 cycles under standard conditions using 0.1 µM primer concentrations at an annealing temperature of 55 C. Using these primers, a homologous recombination event resulted in amplification of a 2.3-kb fragment. Additional reactions were run on all DNA samples using the same upstream primer and a downstream primer to sequences in exon 3 of the BP-2 gene (5'-CAGGGAGTAGAGATGTTCCA-3') that were removed in the construction of the targeted null allele. Amplification with these primers detected a 1.5-kb band from the normal endogenous BP-2 allele.

Genotypes of putative positive ES clones and subsequent progeny from chimera matings were confirmed by Southern analysis. Purified ES and tail DNAs were digested with EcoRI, phenol chloroform extracted, ethanol precipitated, and separated by agarose gel electrophoresis. DNAs were then denatured, blotted onto Genescreen nylon membranes (NEN Life Science Products, Boston, MA) and used for hybridization with a random-primed cDNA probe digested from the parent plasmid with HincII so that it included only exons 2, 3, and 4 of rat IGFBP-2 (80). Hybridizations were performed overnight at 42 C in 50% (vol/vol) deionized formamide, 2xSSC (1xSSC contains 0.15 M NaCl and 0.015 M sodium citrate), 1% SDS, 10% dextran sulfate, 250 µg/ml denatured herring sperm DNA, and 1–3 x 106 cpm/ml of a [{alpha}-32P]CTP-labeled cDNA probe to rat IGFBP-2 (80). After hybridization, blots were washed twice at 65 C in 2xSSC, 1% SDS followed by two washes in 0.2xSSC, 1% SDS and exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -80 C with an intensifying screen.

RNA Analysis
Total cellular RNA was extracted and purified as described by Chomczynski et al. (81). For Northern analysis, RNA samples were separated by electrophoresis through 1% agarose gels containing 2.2 M formaldehyde and transferred to Genescreen nylon membrane. Hybridizations were performed at 42 C in 50% (vol/vol) deionized formamide, 5x Denhardts (1x Denhardts contains 0.02% Ficoll, 0.02% polyvinylpyrrolidine, and 0.02% BSA), 5xSSPE (1xSSPE contains 0.15 NACL, 0.01 [SCAP]M sodium phosphate and 0.001 M EDTA), 1% SDS, 100 µg/ml denatured herring sperm DNA, and 1–3 x 106 cpm/ml of a [{alpha}-32P]CTP-labeled cDNA probe to rat IGFBP-2 (80) or to rat ß-actin (provided by J. Roberts). The cDNA to rat ß-actin encoded exons 1–2 and was derived from a full-length rat cDNA sequence (82). The cDNA inserts were labeled by random priming and had a specific activity of 109 cpm/ml (Roche Molecular Biochemicals, Indianapolis, IN). Membranes were washed twice for 15 min in 2xSSPE, 1% SDS at 65 C followed by twice for10 min in 0.2xSSPE, 1% SDS at 50 C and exposed to Kodak XAR-5 film using an intensifying screen at -80 C.

Total head and trunk RNAs isolated from e13.5 and e17 BP-2 heterozygous or homozygous embryos were used for ribonuclease protection assays as previously described (83). Each RNA sample (5 µg) was hybridized simultaneously with 32P-labeled RNA antisense probes to mouse BP-5 and mouse ß-actin. Probes were transcribed from linearized plasmids containing mouse BP-5 (19) or actin cDNAs (provided by A. Efstratiadis). Total counts per min in each protected band was determined by scintillation counting of excised bands. Subsequent values for BP-5 were corrected based on values for the actin band in each sample to normalize for variation between samples.

Ligand and Immunoblotting
One to 3 µl serum from each animal was run on 12.5% polyacrylamide gels using either the Mini-Protean II or the Protean IIxi system (Bio-Rad Laboratories, Inc., Richmond, CA). For full size gels, samples were electrophoresed through 16–17 cm resolving gels that were run at 85 V for 12–14 h. For minigels, samples were run through 5–6 cm gels at 85 V for approximately 2 h.

For all ligand blots, proteins were transferred to a polyvinylidene fluoride (PVDF) nylon membrane (Millipore Corp., Bedford, MA) which provided a signal intensity equivalent to that observed after transfer to nitrocellulose. Membranes were preblocked with 1% BSA in Tris-buffered saline (TBS) containing 3% NP-40 and 0.1% Tween-20 before overnight incubation with 400,000 cpm of 125I-IGF-I or 125I-IGF-II at 4 C. After sequential washes in TBS containing 0.1% Tween-20 and in TBS alone, blots were exposed to Kodak XAR-5 film for varying durations ranging from 1 day to 3 weeks.

For immunoblots, serum samples were electrophoresed as for ligand blotting and electroblotted onto either nitrocellulose or PVDF membranes. The membrane was preblocked with blotto (5% dry milk in TBS) and incubated overnight at 4 C in antiserum against either rat IGFBP-2 (1:1000; antisera provided by M. Rechler)(80) or rat IGFBP-3 (1:500; provided by N. Ling) (84). After washes in TBS and TBS plus 0.5% Tween-20, blots were incubated in secondary antibody (AP-conjugated Anti-Rabbit IgG; Promega Corp.) for 1 h and washed again in TBS and TBS plus 0.5% Tween-20. Blots were then visualized by treatment with BCIP(X-phos) and nitroblue tetrazolium (NBT) in 0.1 M Tris (pH 9.5), 0.1 M NaCl, and 50 mM MgCl2.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. E. Robertson for providing the CCE embryonic stem cells, Drs. M. Rechler, N. Ling, and D. Clemmons for providing IGFBP antibodies, Drs. M. Rechler, P. Rotwein, J. Roberts, and A. Efstratiadis for providing cDNA clones, and Dr. S. Potter for providing the gene targeting vector. The authors would also like to thank Dr. R. Bonneau for assistance with spleen analyses and Paul Hsu, Kristin Concepcion, Carolyn Wahl, and Jennifer Thompson for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: John E. Pintar, Department of Neurobiology and Cell Biology (J.E.P.), Robert Wood Johnson Medical School, University of Medicine and Dentistry New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854.

This work was supported in part by NIH Grant NS-21970 to J.E.P. and NIH Grant DK-48103 to T.L.W.

1 Present address: Department of Neuroscience and Anatomy, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033. Back

2 Present address: Liver Research Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461. Back

Received for publication October 7, 1999. Revision received May 18, 2000. Accepted for publication May 30, 2000.


    REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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