ABSTRACT
Aim To construct and identify a new time-specific NLRP3 point mutation transgenic mouse model by Cre-LoxP system. Methods Cre-LoxP system was used to generate NL-RP3
ABSTRACT
AIM: To construct and identify the mammary gland cell-specific conditional knockout of SENP7 by the Cre-loxP system. METHODS: The homozygous SENP7
ABSTRACT
Objective@#To construct and identify a mouse model with conditional knockout (cKO) of p75 neurotrophin receptor (p75NTR-cKO) gene in epidermis cells by Cre-loxP system.@*Methods@#Five p75NTRflox/flox transgenic C57BL/6J mice (aged 6-8 weeks, male and female unlimited, the age and sex of mice used for reproduction were the same below) and five keratin 14 promotor-driven (KRT14-) Cre+ /- transgenic C57BL/6J mice were bred and hybridized via Cre-loxP system. Five p75NTRflox/+ ·KRT14-Cre+ /- mice selected from the first generation of mice were mated with five p75NTRflox/flox mice to obtain the second generation hybrids. After the second generation mice were born 20-25 days, the parts of the mice tail were cut off to identify the genotype by polymerase chain reaction method. Four p75NTR gene complete cKO mice (6 weeks old) and 4 wild-type mice (6 weeks old) were selected and sacrificed respectively. The abdominal skin tissue and brain tissue were excised to observe the expression of p75NTR in the two tissue of two types of mice by immunohistochemical staining. The abdominal skin tissue of two types of mice was obtained to observe the histomorphological changes by hematoxylin and eosin staining.@*Results@#(1) Twenty second generation mice were bred. The genotype of 4 mice was p75NTRflox/flox·KRT14-Cre+ /-(p75NTR-/-), i. e. p75NTR gene complete cKO mice; the genotype of 5 mice was p75NTRflox/+ ·KRT14-Cre+ /-, i. e. p75NTR gene partial cKO mice; the genotype of 5 mice was p75NTRflox/flox·KRT14-Cre-/-, and that of 6 mice was p75NTRflox/+ ·KRT14-Cre-/-, all of which were wild-type mice. (2) The expression of p75NTR was negative in skin epidermis tissue of p75NTR gene complete cKO mice, while numerous p75NTR positive expression was observed in skin epidermis tissue of wild-type mice. Abundant p75NTR positive expression was observed in brain tissue of both wild-type mice and p75NTR gene complete cKO mice. (3) There was no abnormal growth of skin epidermis tissue in both wild-type mice and p75NTR gene complete cKO mice, with intact hair follicle structure.@*Conclusions@#Applying Cre-loxP system can successfully construct a p75NTR-cKO mice model in epidermis cells without obvious changes in skin histomorphology.
ABSTRACT
Genetically engineered mouse models are commonly preferred for studying the human disease due to genetic and pathophysiological similarities between mice and humans. In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner. A various tissue-specific Cre-driver mouse lines have been generated to date, and new Cre lines are still being developed. This review provides a brief overview of Cre-loxP system and a few commonly used promoters for expression of tissue-specific Cre recombinase. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization.
Subject(s)
Animals , Humans , Mice , RecombinasesABSTRACT
Objective To investigate the role of Glial cells missing 2 (Gcm2) in pathogenesis of hypoparathyroidism by knocking out Gcm2 gene in adult mice.Methods Tamoxifen was used to induce conditional knock-out of Gcm2 gene in Gcm2E2fl/flCre-ER mice.Genotypes of knock-out mice were identified by PCR.The protein expression level of Gcm2 was measured by Western blotting.The serum calcium and phosphorus were detected by the calcium and phosphorus assay kits, and the serum parathyroid hormone (PTH) level was detected by ELISA.Parathyroid cell proliferation was tested by Ki-67 immunohistochemical assay.The mRNA expression levels of PTH and calcium sensing receptor (CaSR) were detected by Real-time PCR.Bone mineral density was detected by micro CT.Results Gcm2 gene of parathyroid was confirmed to be knocked out by PCR.Compared with wild type and solvent control groups, Gcm2 knock-out group showed markedly lower protein expression of Gcm2, notably higher serum phosphorus and lower serum calcium and PTH concentrations (all P<0.01).The proliferation of parathyroid cells in Gcm2 knock-out mice were significantly higher(both P<0.01).The mRNA levels of PTH and CaSR in parathyroid gland of the knock-out group were significantly reduced (all P<0.01).Bone mineral density was significantly higher in Gcm2 knock-out group (all P<0.01).Conclusion Knockout of Gcm2 can lead to hypoparathyroidism in adult mice, indicating that Gcm2 is probably a therapeutic target for hypoparathyroidism.
ABSTRACT
Genetically engineered mice have provided much information about gene function in the field of developmental biology. Recently, conditional gene targeting using the Cre/loxP system has been developed to control the cell type and timing of the target gene expression. The increase in number of kidney-specific Cre mice allows for the analysis of phenotypes that cannot be addressed by conventional gene targeting. The mammalian kidney is a vital organ that plays a critical homeostatic role in the regulation of body fluid composition and excretion of waste products. The interactions between epithelial and mesenchymal cells are very critical events in the field of developmental biology, especially renal development. Kidney development is a complex process, requiring inductive interactions between epithelial and mesenchymal cells that eventually lead to the growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. Through the use of genetically engineered mouse models, the molecular bases for many of the events in the developing kidney have been identified. Defective morphogenesis may result in clinical phenotypes that range from complete renal agenesis to diseases such as hypertension that exist in the setting of grossly normal kidneys. In this review, we focus on the growth and transcription factors that define kidney progenitor cell populations, initiate ureteric bud branching, induce nephron formation within the metanephric mesenchyme, and differentiate stromal and vascular progenitors in the metanephric mesenchyme.
Subject(s)
Animals , Mice , Body Fluids , Congenital Abnormalities , Developmental Biology , Epithelial Cells , Gene Expression , Gene Targeting , Hypertension , Kidney , Kidney Diseases , Mesoderm , Morphogenesis , Nephrons , Phenotype , Stem Cells , Transcription Factors , Ureter , Waste ProductsABSTRACT
Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.
ABSTRACT
This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0-plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T0-plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.