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Very long chain polyunsaturated fatty acids (VLC-PUFAs) are unique fatty acids in tissues of mammals such as retina and testis, and the key enzyme of its biosynthesis is very long chain fatty acid elongase 4 (Elovl4). Development of an animal model of tissue-specific knockout of Elovl4 gene is conducive to the in-depth study of the biological function of VLC-PUFAs. Therefore, we constructed Stra8-Cre mice and Elovl4 floxed mice based on Cre / loxP system, and obtained the ( Elovl4 [ flox /+], Stra8-Cre ) heterozygous knockout mice by hybridization. Subsequently, female mice were selected to cross with male mice with homozygous Elovl4 [ flox / flox ] to gain homozygous mice ( Elovl4 [ flox / flox ], Stra8-Cre ) through genotype identification and screening. RT-PCR, qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence techniques were used to detect the knock-out efficiency of Elovl4 in testis. The expression of Elovl4 in testis of both heterozygous and homozygous knockout mice were significantly down-regulated at mRNA and protein levels, but were not affected in other tissues. In summary, we constructed a mouse model with specific knockout of Elovl4 gene in testis, which provides a reliable animal model for studying the effect of VLC-PUFAs on the reproductive function of male mice and the underpinning molecular mechanisms.

Objective To study the process and significance of intercellular mRNA transfer by using a Cre-loxP system. Methods Cre-expressing donor cells and loxP-DsRed-loxP-eGFP-expressing recipient cells were established. Co-culture and Transwell TM co-culture of both donor and recipient cells were analyzed with a separate culture of recipient cells only as controls. After 72 hours, the eGFP + recipient cells in each group were observed and analyzed. Immunofluorescence staining was used to detect Cre expression in eGFP + cells. The effects of donor cell death, cell membrane nanotubes, and ROCK signaling pathway on Cre mRNA transfer were explored. Results After co-culture for 72 hours, the eGFP + cells were only observed in co-culture group, but not in the recipient cells alone group and the Transwell TM co-culture group, indicating that cell-cell contact could mediate Cre mRNA transfer. Immunofluorescence staining showed that Cre mRNA could be translated into proteins and transported to the nucleus in eGFP + recipient cells. Moreover, the transport of Cre mRNA between living cells could be partially blocked by membrane nanotubes or ROCK signaling pathway inhibitors. Conclusion The mRNA can be exchanged among living cells through intercellular contact, thus leading to the changes of biological functions in the recipient cells.

Objective: To explore the effect of γ-aminobutyric acid (GABA)ergic neuronal circuit of the central amygdaloid nucleus (CeA) and the paraventricular nucleus of hypothalamus (PVN) on electroacupuncture (EA)-induced regulation of gastric function by way of CeA-PVN projection., Methods: The present study included 3 parts: 1) C57BL/6 mice were randomly divided into control and EA groups ( n =6 in each group). EA was applied to right "Weishu"(BL21, Back-shu point) and "Zhongwan"(CV12, Front-mu point) for 20 min, followed by detecting the expression of c-fos in the CeA and PVN by using immunofluorescence staining; 2) Microinjection of anterograde tracer (rAAV-EF1α-DIO-mcherry-WPRE-pA) into the CeA was conducted in GAD2-Cre mice for confirming the projection of GABAergic neurons from CeA to PVN; 3) GAD2-Cre mice were randomly divided into rAAV-DIO-mcherry (intra-CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA), rAAV-DIO-hM3D（Gq）-mcherry(intra-CeA injection of rAAV-EF1α-DIO-hM3D(Gq)-mcherry-WPRE-pA) and rAAV-DIO-hM3D（Gq）-mcherry+EA groups( n =6 in each group). The food intake and gastric empty were detected, and the concentration of GABA in the PVN was assayed by using high performance liquid chromatography on the 28 th day after intra-CeA injection., Results: 1) The expression of c-fos in the CeA and PVN was significantly increased in the EA group relevant to the control group( P <0.01), suggesting an activation of neurons in both CeA and PVN after EA. 2) Following CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA, the densely expressed virus GABAergic neurons were found in CeA and large number of projection fibers found in the PVN, suggesting a direct connection between CeA and PVN. 3) After activating the GABAergic neurons of CeA, the concentration of GABA in the PVN was obviously increased ( P <0.01), the food intake and the gastric empty were considerably decreased relevant to the rAAV-DIO-mcherry group( P <0.01). Following EA intervention，the concentration of GABA in the PVN was obviously decreased( P <0.01), the food intake and the gastric empty were significantly increased relevant to the rAAV-DIO-hM3D（Gq）-mcherry group ( P <0.01)., Conclusion: EA of BL21 and CV12 (Back-shu and Front-mu acupoints) can increase food intake and gastric empty in GAD2-Cre mice, which may be achieved by suppressing the release of GABA in PVN through CeA-PVN GABAergic neural circuit.

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 p75NTR(flox/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 p75NTR(flox/+) ·KRT14-Cre(+ /-) mice selected from the first generation of mice were mated with five p75NTR(flox/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 p75NTR(flox/flox)·KRT14-Cre(+ /-)(p75NTR(-/-)), i. e. p75NTR gene complete cKO mice; the genotype of 5 mice was p75NTR(flox/+) ·KRT14-Cre(+ /-), i. e. p75NTR gene partial cKO mice; the genotype of 5 mice was p75NTR(flox/flox)·KRT14-Cre(-/-), and that of 6 mice was p75NTR(flox/+) ·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.

Cre-lox recombination system consists of two elements: Cre recombinase enzyme and lox sites. Cre recombinase can recombine the lox site sequences by specifically detecting and cutting them. The direction and position of lox sites determine the functional effects of Cre enzyme such as deletion, inversion or chromosomal translocation. The hematopoietic system of mouse consists of multi-lineages and various developmental stage hematopoietic cells that are differentiated from hematopoietic stem cells (hematopoietic stem cells, HSC). The hematopoietic stem cells are maintained in the bone marrow microenvironment (niche). Currently, a variety of floxed conditional-knockout mice, recognized by Cre-lox recombination system, are used for the study of the hematopoietic system. This review summarizes the commonly used Cre transgenic mice and their applications in the study of hematopoietic system.

Cell lineage tracing is to track certain cell and all of its progeny. From the 20th century, lineage tracing has provided a predominant method to study organ development, tissue repairiaed cell fate-determination. Recently, the rapid development of technique for gene engineering, especially the application of Cre/loxp recombinase system expands the application range of lineage tracing. Here we discuss the application of lineage tracing technique in different studies, and summarize the characteristics and recent progresses of this technique.

Marker-free plants have been public concern. Co-transformation and site-specific recombination system are more important methods in self-gene excision. We reviewed the Cre/lox site-specific system and its applications in plants, also, we discussed perspectives of the system in according with our experience.