cTAGE5 mediates collagen secretion through interaction with TANGO1 at endoplasmic reticulum exit sites

Cutaneous T-cell lymphoma-associated antigen 5 (cTAGE5), an originally identified tumor antigen, is overexpressed in various cancer cell lines. The cDNA encodes an integral membrane protein containing two coiled-coil motifs and a proline-rich domain. We show that cTAGE5 specifically localizes to the endoplasmic reticulum (ER) exit sites. In addition, cTAGE5 forms a complex with TANGO1 (MIA3), a previously characterized cargo receptor for collagen VII, by the interaction of their coiled-coil motifs. Of interest, cTAGE5, as well as TANGO1, is capable of interacting with the inner-layer coatomer of COPII Sec23/24 complex through their C-terminal proline-rich domains and required for collagen VII secretion. We propose that cTAGE5 acts as a coreceptor of TANGO1 for collagen VII export from the ER.     

Disruption of proprotein convertase 1/3 (PC1/3) expression in mice causes innate immune defects and uncontrolled cytokine secretion

The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.     

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.     

Of mice and MEN1: Insulinomas in a conditional mouse knockout

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.     

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Neural and orofacial defects in Folp1 knockout mice [corrected

BACKGROUND

Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid‐binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax‐3, En‐2, Hox‐a1, Shh, Bmp‐4, Wnt‐1, and Pax‐1).

METHODS

Folbp1 mice were supplemented with low dosages of folinic acid to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization.

RESULTS

In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax‐3 and En‐2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax‐3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax‐1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp‐4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules.

CONCLUSIONS

Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis‐expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice. Birth Defects Research (Part A) 67:209–218, 2003. © 2003 Wiley‐Liss, Inc.

     

CRISPR/Cas9敲除PLIN1基因增强3T3-L1脂肪细胞的脂解作用

应用CRISPR/Cas9技术敲除3T3-L1前脂肪细胞plin1,观察PLIN1缺失对脂肪细胞中脂肪水解的影响并探究可能机制。常规培养3T3-L1前脂肪细胞,电穿孔法转染plin1敲除载体,嘌呤霉素培养基挑选plin1敲除细胞,观察转染及筛选后的细胞存活率。"鸡尾酒"法诱导3T3-L1前脂肪细胞分化,酶法测定甘油和TG含量,油红O染色观察脂滴形态及数目的变化。Western blotting检测PLIN1、PPARγ、Fsp27和脂肪酶的蛋白表达;RT-PCR检测PLIN1和脂肪酶的mRNA表达。对照组细胞诱导分化后,微小脂滴数目较少,单房脂滴数目较多并围绕细胞核呈环型排列。相较于对照组,敲除组细胞诱导分化后微小脂滴数目增加,单房脂滴体积缩小,数目减少;细胞中PLIN1mRNA及蛋白表达被显著抑制(P0.05);甘油水平显著上升(0.0984±0.0076),TG含量显著下降(0.031 0±0.005 3);HSL和ATGL两种脂肪酶的mRNA及蛋白表达均升高(P0.05);PPARγ和Fsp27的表达未有明显变化。上述结果表明plin1敲除后通过暴露脂滴中脂质以及上调脂肪酶等效应增强了3T3-L1脂肪细胞的脂解作用。     

Caveolin-1 knockout alters beta-adrenoceptors function in mouse small intestine

beta-Adrenoceptors are G protein-coupled receptors whose functions are closely associated with caveolae in the heart and cultured cell lines. In the gut, they are responsible, at least in part, for the mediation of the sympathetic stimulation that might lead to intestinal paralysis postoperatively. We examined the effect of caveolin-1 knockout on the beta-adrenoceptor response in mouse small intestine. The relaxation response to (-)-isoprenaline in carbachol-contracted small intestinal tissue segments was reduced in caveolin-1 knockout mice (cav1(-/-)) compared with their genetic controls (cav1(+/+)). Immunohistochemical staining showed that beta-adrenoceptor expression was similar in both strains in gut smooth muscle. Selective beta-adrenoceptor blockers shifted the concentration response curve (CRC) of (-)-isoprenaline to the right in cav1(+/+) intestine, but not in cav1(-/-), with greatest shift in case of the beta(3)-blocker, SR59230A. The CRC of the selective beta(3)-agonist BRL 37344 was also shifted to the right in cav1(-/-) compared with cav1(+/+). The cAMP-dependent protein kinase (PKA) inhibitor H-89 shifted the CRC of (-)-isoprenaline to the right in cav1(+/+) but not in cav1(-/-). H-89 reduced the relaxation due to forskolin and dibutyryl cAMP in cav1(+/+) but not in cav1(-/-), suggesting a reduction in PKA activity in cav1(-/-). In cav1(+/+), PKA was colocalized with caveolin-1 in the cell membrane, but PKA immunoreactivity persisted in cav1(-/-). Examination of PKA expression in the lipid raft-rich membrane fraction of the jejunum revealed reduced PKA expression in cav1(-/-) compared with cav1(+/+). The results of the present study show that the function of beta-adrenoceptors is reduced in cav1(-/-) small intestine likely owing to reduced PKA activity.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

Double knockout Nme1/Nme2 mouse model suggests a critical role for NDP kinases in erythroid development

Nm23/NDP kinases A and B encoded by the Nme1/Nme2 genes are multifunctional enzymes responsible for the majority of NDP kinase activity in mammals. This review summarizes recent studies on their physiological roles using a mouse model in which both Nme1 and Nme2 genes have been deleted. The double knockout mice are stunted in growth and die perinatally. Additionally, these mice display hematologic phenotypes, including severe anemia, abnormal erythroid cell development, loss of the iron transport receptor molecule TfR1, and reduced iron uptake by Nme1 ^?/? /Nme2 ^?/? erythroid cells. We hypothesize that Nm23/NDP kinases regulate TfR1 gene expression in erythroid cells in some manner, and that defective iron transport into these cells is responsible for the anemia and death. This Nme1/Nme2 mouse model also links nucleotide metabolism with erythropoiesis, suggesting alternative or additional mechanisms that may explain the observed phenomena.     

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1–101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.     

The role of hydroxylation in the secretion of collagen by mouse fibroblasts in culture

The secretion of collagen may be specifically and quantitatively measured in a cell culture system using mouse fibroblasts, line 3T6. The results of such a study show that inhibition of the hydroxylation of collagen with α,α′-dipyridyl reduces the rate of secretion to 27% of the rate of normally hydroxylated collagen. Dipyridyl does not hinder passage of the normally hydroxylated collagen species to the medium. Therefore its effect on the secretion of under-hydroxylated collagen (protocollagen) is due to its inhibition of hydroxylation and not to an inhibition of the cell's secretory machinery.     

Association between endothelin-1 and collagen deposition in db/db diabetic mouse kidneys

Endothelin-1 has been implicated in diabetic kidney injury, but there are few firm data establishing the temporal and spatial expression of kidney endothelin-1 in diabetes. We performed an immunohistochemical and histopathological analysis to determine endothelin-1 peptide expression in the kidneys of diabetic db/db mice and non-diabetic db/m controls. Diabetic mice were studied at 8 weeks, before histological damage is evident, and again at 16 weeks, when significant glomerular injury has occurred. Urinary endothelin-1 was 6.2- and 3.6-fold higher in 8- and 16-week diabetic mice compared to age-matched controls (P<0.01 db/db vs. db/m). Compared to non-diabetic kidneys, immunoreactive endothelin-1 was first elevated 2.5-fold (P=0.02) in the tubulointerstitial compartment at 8-week and remained high (3.8-fold, P<0.01) at 16 weeks. In contrast, glomerular endothelin-1 was elevated 3.2-fold (P=0.03) only in 16-week diabetic mice. Glomerular and tubulointerstitial endothelin-1 were unchanged in 8- and 16-week non-diabetic mice. Elevated endothelin-1 in diabetic mice associated temporally and spatially with collagen deposition, especially in the tubulointerstitial compartment. The localization of kidney endothelin-1 is consistent with a role for this peptide in renal fibrogenesis. These results also highlight the potential role of ET-1 in the pathogenesis of early tubulointerstitial changes in diabetes.     

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.     

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24 h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system

Insulin-like growth factor-1 (IGF-1) is an essential growth factor for normal intrauterine development and postnatal growth. Mice with a complete deficiency of IGF-1 (IGF-1-null mice), created by homologous recombination, were found to exhibit postnatal lethality, growth retardation, infertility, and profound defects in the development of major organ systems. Furthermore, IGF-1-null mice were resistant to growth hormone (GH) treatment in peri-pubertal somatic growth. Using the Cre/loxP-induced conditional knockout system, we generated a mouse that lacks IGF-1 specifically in the liver, the primary site of IGF-1 production. Interestingly, although circulating and serum levels of IGF-1 were decreased by approximately 75% in these mice, they exhibited no defect in growth or development. When administered exogenously, GH stimulated IGF-1 production in several extra-hepatic tissues as well as body growth. The "Somatomedin hypothesis" originally proposed that circulating IGF-1 acting in various tissues mediate the effects of GH. These striking in vivo results, obtained using homologous recombination technology, call for a major modification of the Somatomedin hypothesis. These gene targeting studies confirm that IGF-1 is essential for GH-stimulated postnatal body growth. However, liver-derived (endocrine) IGF-1 is not essential for normal postnatal growth, though it does exert a negative feedback on GH secretion. Instead, local production of IGF-1, acting in a paracrine/autocrine fashion, appears to mediate GH-induced somatic growth. This review will discuss the effects of tissue-specific IGF-1 gene deficiency created by the Cre/loxP system versus the conventional IGF-1 knockout.