High-bone-turnover Osteoporosis and Aortic Calcification in Opg Knockout Mice / 生物化学与生物物理进展

Bone turnover is regulated by local concentrations of cytokines such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL). To explore the in vivo biological function of Opg and the mechanism of osteoporosis due to deficiency of Opg, Opg knockout mice have been generated through homologous recombination. Opg-/- mice exhibit a sharply decrease in bone density and strength as expected. The number of osteoclasts in Opg-/- mice significantly increases. Morphologically, osteoclasts appear more cuboidal in shape in Opg-/- mice than those of wt mice, suggesting that active osteoclastogenesis occurs in the absence of Opg. In consistent with this finding, an increase of osteoblast activity was also observed with accelerated mineral accumulation rate by histomorphometric measurement and elevated serum alkaline phosphatase activity (ALP) in Opg-/- mice. Interestingly, more than 50% of 2-month-old Opg-/- mice manifest medial calcification of aorta with comparable serum concentrations of calcium and phosphorus to wt mice. In conclusion, Opg-/- mice have a high-bone-rurnover type osteoporosis. The aortic calcification in Opg-/- mice is not due to abnormality of calcium and phosphorus metabolism. The mechanism underlying aortic calcification in Opg-/- mice needs to be further investigated.

Generation of Adiponectin Gene Knock-out and LacZ Gene Knock-in Mouse Model / 生物化学与生物物理进展

Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.

Conditional targeting of p16(INK4a)exon 1a in mouse embryonic stem cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16(INK4a) plays a role in tumor suppression.</p><p><b>METHODS</b>A conditional targeting vector with 2.0 kb EcoR I/Xba I fragment as short arm and 5.9 kb SpeI/NotI fragment as long arm was built. Of the 2 direct locus crossing- over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16(INK4a) exon 1a and the other at 1633 bp downstream of the initiate code. Both exon 1a and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, t he targeting vector was introduced into ES cells through electroporation.</p><p><b>RESULTS</b>Twenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization.</p><p><b>CONCLUSION</b>Targeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.</p>

Analysis of the factors effecting the expression efficiency of the green fluorescent protein gene in mouse embryonic stem cells / 第二军医大学学报

Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.

Biological properties of the mouse embryonic stem cell line expressing green fluorescent protein / 第二军医大学学报

Objective: To study the characteristics of mouse embryonic stem (ES) cell line R1 expressing green fluorescent protein (GFP) efficiently and stably. Methods: Four kinds of biological characteristics: growth curve, express level of alkaline phosphatase, karyotype and pluripotentiality of the subclones of R1 ES cell expressing GFP were observed and analysed. Results: No obvious differences between R1 ES cells and the 4 ES cell clones expressing GFP［ESG（+）］ on the proliferation speed, differentiation state and the ratio of normal karyotype were observed. Teratocarcinoma concluded 3 germinal layers could form after inoculating ESG(+) cells to nude mice. Conclusion: The expression of GFP may not make any detectable effect on the proliferation speed, differentiation state and the pluripotentiality of R1 ES cells. This research work ensures efficient utilization of GFP as a reporter gene tracing ES cell in vivo.

A dynamic study of p53 gene mutations in the development of colorectal adenoma-carcinoma sequence / 中华消化杂志

p53 gene mutations and their expression in protein levels of colorectal adenomas, carcinomas and regional lymph nodes metastasis were detected by PCR-SSCP and AB-PAP immunohistochemical analysis, respectively. The results showed that p53 gene mutations were detected in 25% colorectal adenomas, 80% carcinomas and 100% regional lymph nodes metastasis. Meanwhile, p53 proteins were detected at rates of 75%, 60% and 57% in these three groups, respectively. It is suggested that p53 gene mutations occur frequently at late stage of progression to colorectal adenoma-carcinoma sequence and may relate to its metastasis, while the accumulation of p53 protein may be a specific genetic alteration initiated from adenomatous stage. Thus, the biological significances of the two genetic alterations are different. Investigations of these concomitantly would be helpful in understanding the malignant potential of adenoma and evaluation of its staging.

Construction and application of a mouse HPRT targeting vector with Cre recombinase recognition site lox66 / 第二军医大学学报

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.

Development of pMTR1 plasmid-based transgenic mice model for studying gene mutation in vivo / 第二军医大学学报

Objective: To establish C57BL/6 transgenic mice lineages containing pMTR1 plasmids as an efficient animal model for studying gene mutation in vivo . Methods: The linearized pMTR1 DNAs, constructed by molecular cloning, were injected into 572 fertilized eggs of C57BL/6 mice by microinjection. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant mice respectively, from which 35 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Four mice whose genomes integrated with copies of intact pMTR1 vectors were chosen as founders to establish transgenic mice lineages. The recovery of pMTR1 plasmid were conducted on the genomic DNAs of F1 or F2 transgenic offsprings. Results: It was showed that intact and functional pMTR1 plasmid could be efficiently recovered from the genomic DNAs of these mice. Conclusion: (1) The transgenic mouse lineages containing copies of a stably integrated pMTR1 plasmid is established. (2) The transgenic mice are suitable models for studying gene mutation in vivo .

Analysis of genetic structure and gene flow of HLA class Ⅱ in 11 groups of the Chinese Nation / 第二军医大学学报

Objective: To ascertain the gene flow tracks and patterns and the origin of HLA class Ⅱ in the Chinese Nation.Methods : HLA classⅡ in 11 Chinese groups and 5 reference populations was studied by clustering analysis of genetic structure and distribution comparison of gene frequencies.Results and Conclusion: These analyses revealed that these genes decreased southwards and originated probably in Caucasoid or Negroids (DQA 1*0501, DQB1*0501, etc.),and those decreased northwards and originated probably in Southeast Asia (DQA1* 0601, DRB1*1202, etc.).It was found that these genes decreased both southwards and northwards (DQB1*0303, DRB1*0901,*1501,etc.) and originated probably in certain groups in mainland of Chinese Nation,and that HLA genes in different Chinese groups migrated from each other and amalgamated deeply,but Dais, Buyis etc were isolated from outside in some degree. Our results also supported that the Chinese Nation comprises two major populations of north and south.We proposed that the migration and mixing are the dominant driving force of the HLA gene flow while the genetic drift caused by isolation and the nature selection have an important influence on HLA genetic structure.

Influence of expression of splice variants of CD44 in tumor-adjacent tissue upon the recurrence of primary liver cancer after operation / 第二军医大学学报

To study the significance of the expression of splice variants of CD44 (CD44v) in tu-m0r-adjacent tissue of the patients with primary liver cancer(PLC). Methods: To research into the signifi-cance of the expression of CD44v mRNA in tumor-adjacent tissue of 30 patients with PLC by RT-PCR andfollow-up. Results: In the patients that the expression of CD44v mRNA of tumor-adjacent tissue washigher than those of tumor tissue(group I ), clinical pathological indexes were higher than in the patientsthat the expression of CD44v mRNA was higher than the tumor tissue(group I ). The recurrent rate ofgroup I was higher than that of group n (P