Chunfang Zhang1,*, Danny Kitsberg2, Hun Chy1, Qi Zhou2 and John R. Morrison1,3
1 CopyRat Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 2 IngenKO Pty Ltd 27-31 Wright Street, Clayton, Victoria 3168, Australia 3 Monash Institute of Reproduction and Development, Monash University 27-31 Wright Street, Clayton, Victoria 3168, Australia
Received October 31, 2004. Revised December 22, 2004. Accepted December 22, 2004.
Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.
4.Endocrinological abnormalities in angiotensinogen-gene knockout mice: studies of hormonal responses to dietary salt loading.
Journal of Hypertension. 16(3):285-289, March 1998.
Umemura, Satoshi 1,3; Kihara, Minoru 1; Sumida, Youichi 1; Yabana, Machiko 1; Ishigami, Tomoaki 1; Tamura, Kouichi 1; Nyui, Nobuo 1; Hibi, Kiyoshi 1; Murakami, Kazuo 2; Fukamizu, Akiyoshi 2; Ishii, Masao 1
Abstract:
Objective: Physiological roles of the renin-angiotensin system in maintaining blood pressure and sodium-water balance in angiotensinogen gene-knockout mice were evaluated with special reference to endogenous pressor substances.
Methods: Angiotensinogen-gene knockout mice and control mice were fed a 0.3 or 4% NaCl diet for 2 weeks. Systolic blood pressure and urinary excretions of electrolytes, creatinine, aldosterone, adrenaline, noradrenaline, dopamine and vasopressin were measured.
Results: About 60% of our angiotensinogen-gene knockout mice did not survive until weaning. These mice presented with hypotension and polyuria. Urinary excretion of aldosterone from such mice was significantly lower (not detected) than that from control mice (2.0 +/- 0.3 pg/mg creatinine). In contrast, urinary excretion of vasopressin from angiotensinogen-gene knockout mice (0.7 +/- 0.1 ng/mg creatinine) was greater than that from control mice (0.3 +/- 0.1 ng/mg creatinine), and those of adrenaline and of noradrenaline were similar for knockout and control mice. After salt loading (a 4% NaCl diet), angiotensinogen-gene knockout mice exhibited a significant increase in systolic blood pressure (from 68.3 +/- 2.9 to 95.9 +/- 5.9 mmHg), significant decreases in urinary excretions of adrenaline (from 65 +/- 8 to 40 +/- 7 pg/mg creatinine) and noradrenaline (from 467 +/- 48 to 281 +/- 41 pg/mg creatinine) and no change in excretion of vasopressin compared with such mice fed a 0.3% NaCl diet.
Conclusion: The present results with angiotensinogen gene knockout mice confirm that the renin-angiotensin system plays fundamental roles in maintaining the blood pressure and sodium-water balance. Because the vasopressin and catecholaminergic systems may be altered by lack of angiotensin in angiotensinogen-gene knockout mice, these systems perhaps are not able to restore blood pressure and sodium-water depletion to normal levels in these mice. J Hypertens 16:285-289 (C) 1998 Rapid Science Ltd.
5.Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock
Salvatore Cuzzocrea*,1, Giuseppe Nocentini, Rosanna Di Paola*, Emanuela Mazzon*, Simona Ronchetti, Tiziana Genovese*, Carmelo Muià*, Achille P. Caputi* and Carlo Riccardi
In the present study, we used glucocorticoid-induced tumor necrosis factor (TNF) receptor family gene knockout (GITR-KO) mice to evaluate a possible role of GITR on the pathogenesis of splanchnic artery occlusion (SAO) shock, which was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum of the SAO-shocked, GITR wild-type (WT) mice after reperfusion. The absence of GITR significantly reduced the lipid peroxidation in the intestine. SAO-shocked WT mice developed a significant increase of ileum tissue, TNF-, and myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (5% survival at 24 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin, intercellular adhesion molecule 1 (ICAM-1), and E-selectin. The intensity and degree of P-selectin, E-selectin, and ICAM-1 were markedly reduced in tissue section from SAO-shocked, GITR-KO mice. SAO-shocked, GITR-KO mice also showed a significant reduction of the TNF- production and neutrophil infiltration into the reperfused intestine, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that GITR plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of GITR expression may represent a novel and possible strategy.
6.Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets
Hal Alper1, Kohei Miyaoku1, 2 & Gregory Stephanopoulos1
Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.
7.Activation of the renin-angiotensin system in [alpha]-calcitonin gene-related peptide/calcitonin gene knockout mice.
Journal of Hypertension. 22(7):1345-1349, July 2004.
Li, Jianping a; Zhao, Huawei a; Supowit, Scott C b; DiPette, Donald J b; Wang, Donna H a
Abstract:
Objective: To test the hypotheses that circulating or tissue renin-angiotensin system (RAS) activity is increased in [alpha]-calcitonin gene-related peptide ([alpha]CGRP) knockout mice, and that this contributes to the increased blood pressure in these mice.
Design and methods: Three- to six-month-old male [alpha]CGRP/calcitonin knockout mice and wild-type controls were studied. Mean arterial pressure (MAP) and its response to an angiotensin II type 1 (AT1) receptor blocker, losartan (3 mg/kg intravenously), were determined in conscious, unrestrained knockout mice and wild-type mice. Radioimmunoassay and western blot were used, respectively, to determine plasma renin activity (PRA) and AT1 receptor protein content in tissues.
Results: Basal MAP and PRA were significantly greater in the knockout mice than in the wild-type mice. In contrast, AT1 receptor content in the renal medulla was significantly decreased in the knockout mice compared with that in wild-type mice. AT1 receptor content in the renal cortex and mesenteric resistance arteries was not different in the knockout and wild-type mice. Losartan produced a significant decrease in MAP in the knockout mice compared with that in wild-type mice.
Conclusion: Activity of the circulating RAS, but not tissue AT1 receptor expression, is increased in [alpha]CGRP/calcitonin knockout mice, which may contribute to the increase in blood pressure in this mouse model. The mechanism(s) responsible for the increased activity of the circulating RAS in the absence of [alpha]CGRP throughout the developmental stages of these animals remains to be determined.
8.Histidine decarboxylase deficiency in gene knockout mice elevates male sex steroid production
E Pap, K Racz, JK Kovacs, I Varga, E Buzas, B Madarasz, C Foldes, C Szalai, T Watanabe, H Ohtsu, A Ichikawa, A Nagy, and A Falus
Histamine is synthesized in cells by histidine decarboxylase (HDC). HDC-deficient knockout (KO) mice lack functional HDC and histamine in the tissues. In the present study we used this in vivo model for studying the role of HDC deficiency in the regulation of male steroid hormone metabolism. In agreement with earlier studies showing the lack of effects of central histamine on the basal secretion of gonadotrope hormones, we found no difference with in situ hybridization in the expression of GnRH in the hypothalamus of wild type and KO mice. The tissue concentrations of testosterone and several androgenic steroids were significantly elevated in the testes but not in the adrenal glands of HDC-KO mice. In contrast, serum estradiol levels failed to show a significant difference between the two groups. The weight of the testes was significantly smaller in both 7-day-old and adult KO mice. The ultrastructure of the adult testis indicated elevated steroid synthesis with more tightly coiled membranous whorls in Leydig cells. The present results suggest that changes in reproductive functions and sex steroid secretion in male HDC-KO mice are not due to altered hypothalamic GnRH expression but are probably related to definite modifications during fetal development of KO mice reinforced later by the lack of the effect of peripheral histamine. This may provide in vivo evidence that peripheral histamine is an important regulatory factor of male gonadal development during embryogenesis and of sex steroid metabolism later in adulthood.
References