OBJECTIVE:To determine AF172993 sequence is either the complete CDS or a transcript variant.METHODS:RT-PCR was used to amplify the CDS sequence of Plunc, which was subsequently cloned into the pEGFP-N1 eukaryotic expression vector. After bi-directional sequence analysis, the sequence obtained was blasted against the AF172993 sequence, nr database and human genome database.RESULTS:In CDS of the new cloned sequence, the 658 base A in the AF172993 sequence was replaced by C, and the corresponding genetic code was also converted from AAG to CAG, leading to the alteration of the amino acid Gln to Lys. In addition, the base C at the 658 position of the CDS showed perfect match with the base C at 2094188 position in human chromosome 20.CONCLUSION:The base A at the 658 position of AF172993 sequence of Plunc is a mutation site, which alters the coding of the amino acid. AF172993 sequence is actually a transcript variant of Plunc, and the annotation to AF172993 in GenBank database is not correct and need to be revised.

OBJECTIVE To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

OBJECTIVE:To study the effects of different parameters for electrofusion on the activation and early development of mouse embryos reconstructed by cumulus cell nuclear transfer, and explore optimal parameters for electrofusion.METHODS:A C57BL/6j mouse cumulus cell nucleus 10-12 mm in diameter was inserted into the perivitelline space of an enucleated oocyte. The fusion of donor-recipient pairs was induced with different parameters for electrofusion (with variation in electric field intensity, pulse duration and pulse times). Successful formation of the reconstructed embryos from donor-recipient pairs and the reconstructed embryos developing into the early embryonic stages (2-cell, 4-8-cell and morula stages) were observed and counted.RESULTS:The electric field intensity and pulse duration allowed variation during electrofusion within the range of 1 000-2 000 kV/cm and 40-160 ms, respectively. The donor-recipient pairs fused at very low rate when the parameters were below the allowed ranges, and disintegration or even death might occur when the parameters were above the ranges. Within these allowed ranges, variation of the electrofusion parameters did not produce significant impact on the ratio of the reconstructed embryos in 2-cell, 4-8-cell or morula stages (P<0.05). In addition, we suggested that pulse times be limited to 1-2.CONCLUSION:Optimal parameters for electrofusion are crucial for the fusion of donor-recipient pairs and the activation of the reconstructed embryo.

In order to elucidate the role of EBV-LMP1 in the nasopharyngeal carcinogenesis, the expression vector was constructed with subjecting the N-LMP1 gene to double regulation of two specific regulators: EDL-2 and PLUNC-p. The N-LMP1 related transgenic mice model has been constructed successfully by pronucleus microinjection. 58 founder mice were born, 4 of which were founded to be positive by PCR and Southern blot. Immunohistochemistry assay showed that N-LMP1 protein was expressed in the nasopharynx, tongue and forestomach of transgenic mice.

The present study examined if injection of DNA into the testis tabular could generate transgenic mice via transfecting spermatogonia. The 0.9 kb Bcl-2 cDNA was fused to the promoter region of mouse whey acid protein gene and the SV40 polyA. A 3.0 kb fragment of WAP-Bcl-2-SV40 was mixed with cationic liposome and one side tabular of 3 mice testis was injected with the fragment, the other side was ligatured. Two out of the 3 males were used to mate with the female 4 days later. Twenty pups were produced and 3 of which were proven to be gene integration positive by PCR detection. Two, 1 male and 1 female, were further confirmed to carry the transgene by Southern blot analysis. The male died by accident during its feeding. The female was demonstrated to express Bcl-2 protein in its mammary glands by Western blot assay. Seven out of 45 F1 mice were proven to be transgenic by PCR. It is concluded that transfecting spermatogonia in vivo can produce transgenic mice.

In present study, transgene LTR-N-LMP1 was microinjec ted into the pron uclei of the mouse zygotes, then these zygotes were transplanted into the pseudo -pregnant female mice. 15 founder mice were obtained. 2 of them were positive f o r gene integration proved by PCR and Southern blot analysis, the positive ratio was 13.3%. These results provided an important basis for exploring N-LMP1's fun ction and mechanism in nasopharyngeal oncogenesis.

Three mammary-specific expression vectors of pCBEA,pWE3′ and pCWEA were constructed with 1.1 kb bovine BLG gene promoter,0.949 kb WAP gene promoter and 2.4 kb erythropoietin genomic DNA minigene.The difference among the three vectors was that WAP3′ UTR was used as termination signal in pWE3′ and bGH polyA in pCBEA,pCWEA and the CMV enhancer element was cloned into the upstream of BLG,WAP gene promoter in pCBEA,pCWEA.The vectors mixed with liposome were injected into mammary glands of late stage pregnant rabbits through mammary gland teat canal respectively.Milk was collected on 1-25 d from rabbits after parturition.The results of ELISA assays showed that the expression level of pCBEA,pWE3′ and pCWEA was at the range of 0.335-0.680 μg/L,0.051-0.516 μg/L and 0.201-0.753 μg/L in the rabbit milk respectively.

To establish nasopharyngeal carcinoma(NPC) derived latent membrane protein 1(N-LMP1) transgenic mice model, and investigate the function of N-LMP1 in vivo, further elucidate the role of Epstein-Barr virus in the process of nasopharyngeal oncogenesis, keratinocyte specific promoter EDL2 was linked to NPC-derived LMP1 by using the DNA recombinant techniques, then transgene EDL2-N-LMP1 was microinjected into the pronuclei of the mouse zygotes, these zygotes were transplanted into the pseudo-pregnant female mice. Expression of N-LMP1 and pathologic change in nasopharyngeal epithelium of founder mice were observed. There were 53 founder mice which were tested by PCR and Southern blot analysis. 6 of them were positive for gene integration, the positive ratio was 11.3%. A typical hyperplasia in nasopharyngeal epithelium were found in one 4-month-old founder mouse. The results suggested transgenic mice bearing N-LMP1 were constructed; N-LMP1 induced atypical hyperplasia of nasopharyngeal epithelium in the mouse, illustrating that the N-LMP1 gene alone might be sufficient to cause the precancerous lesion in mouse nasopharyngeal epithelium. This study provided a strong evidence to verify that EB virus is a key pathogen of NPC.

OBJECTIVE:To study the feasibility of establishing transgenic mice carrying enhanced green flourescent protein (EGFP) gene by means of seminiferous tubule microinjection.METHODS:The vector expressing enhanced green fluorescent protein under the control of human cytomegalovirus immediate-early promoter (pCMV-EGFP) was selected and mixed with liposome in vitro. Microinjection at different doses of the liposome-entrapped plasmid DNA into the seminiferous tubules of male mice at different ages was performed to establish transgenic mice, which were made to mate with female mice at least 40 d after the microinjection. Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis, and the frozen sections of different tissues from 2 of the founders mice were prepared for fluorescence microscopic observation.RESULTS:Among the 41 mice receiving the microinjection, 32 survived and retained their mating ability and fertility, and among their 382 offspring 133 were positive for EGFP DNA as demonstrated by PCR, 15 of which were confirmed by Southern blotting analysis. The age of the mice or the doses of microinjection they received was not shown to impact the integration of EGFP gene, and fluorescence microscopy failed to detect significant EGFP expression in the tissues of the founder mice (P>0.05) in comparison with normal mice.CONCLUSION:Seminiferous tubule microinjection is simple and practicable to implement gene transfer in mice.

Objective To obtain the gene fragment encoding human mannan-binding lectin (MBL)-associated serine protease-2 (MASP-2). Methods The target cDNA fragment was amplified from the total RNA extracted from human fetal liver tissue by RT-nested PCR, cloned into pGEM-T vector and identified by restriction analysis and sequencing. Results The cDNA fragment encoding MASP-2 polypeptide with the signal sequence was isolated, linked with pGEM-T vector and transformed into E.coli TG1. The restriction maps of the selected clones were consistent with those generated by computer analyses. The result of sequencing of one selected clone showed that its sequence was identical with that of reported human MASP-2 cDNA. Conclusion The human MASP-2 gene was successfully cloned, which prepares the ground for studying the molecular mecha- nisms by which MBL activates the complement lectin pathway and the mutations of MBL gene cause immunodeficiency.

Objective To explore the feasibility of sperms used as carriers of foreign gene for generating transgenic mice. Methods The sperms were taken from the vas deferens and ductus epididymidis of mature mice, incubated with foreign plasmid DNA covered by liposome and injected into the vagina of super-ovulated female mice. Forty hours later the 2-cell embryos were collected for test. Results The results showed that: (1) The sperms could be transfected by foreign DNA and the ratio of transfection was 53%; (2) The 2-cell embryos could carry foreign DNA from the sperms transfected and the frequency of carrying was 11.5%. Conclusion Sperms from mouse vas deferens and ductus epididymidis can be transfected by foreign DNA covered by liposome and can be used as the vehicle to carry the foreign DNA into early embryo.

为了获得人促红细胞生成素(EPO)的高效表达,以EPO gDNA 为基础构建了两个真核表达载体pDE、pCE,将它们分别经脂质体转染CHO-dhfr-细胞,其48h瞬时表达量分别为27.5ng/ml与88.90ng/ml.然后用氨甲喋呤(MTX)对转染细胞进行加压并挑选细胞克隆,共获得3株高效表达EPO的工程细胞株A,B,C,其中A来自质粒pCE,B和C来自pDE,在2×10-6mol/L的MTX的压力下,它们48h的最高表达水平分别为A:8.7μg/ml,B:10.76μg/ml,C:16.44μg/ml.经网织红细胞法测得它们均具有体内生物活性.

Two specific expression pasmids, pSVL β ΔWAP PA and pSVL WAPβ PA, containing t PA cDNA gene,were constructed by fusing with cattle β Casein promoter, respectively.The two plasmids were injected directly into pregnant rabbit mammary gland duct. The result showed that both plasmids could express t PA in rabbit milk.The expression level was 700 μg/L and 500 μg/L on day 5, respectively. It provides the useful information for study the regulation of specific expression in mammary gland and the establishment of t PA mammary gland bioreactor.

The transgenic mice of human leukocyte antigen B27 (HLA-B27) has been generated by microinjection of the mixture of 6.5 kb HLA-B27 gene and 15 kb beta_(2)-microglobulin (beta_(2)-m) gene into the fertilized eggs of mice. 1 050 out of 1 370 microinjected eggs have been transplanted to 58 pseudopregnant mice and 97 offspring were produced. Genomic DNA samples from the offspring have been detected by PCR and southern blotting techniques. The results showed that 27 out of 97 mice were positive by analysis of PCR and 8 mice were positive by southern blotting detection.

To achieve high expression of heterologous gene, two universal eukaryotic vectors: pMCD A and pMCD B were constructed from pcDNA3 and pSV2 dhfr. Then EPO minigene were cloned into their multiple cloning sites. The recombinant plasmids were pAE and pBE. They were transfected into CHO dhfr - cells by lipofectin respectively. Their transient expression showed that they can express heterologous gene in CHO dhfr - cells efficiently. By reticulocyte counting, all the secreted EPO revealed their biological activity in vivo.

To find out more simple method for developing transgenic animal, the electroporation was experimented with BAERON 6000 genic transference apparatus. The rabbit sperms were obtained by pseudo vagina method, washed, diluted and mixed with foreign DNA. Then they were electroporated under 9 different conditions, later were digested by DNA 1 enzyme and detected with digoxin in situ hybridization to find out the sperms with internalized foreign DNA. The results showed that electroporation of rabbit sperms could truly lead to internalization of foreign DNA with the optimal condition to be 500 ms of cycle and 6 kV voltage.

Objective To assess the expression ability of WAP-tPA plasmid, which was designed to be expressed specifically in the mammary glands. Method and Results The plasmid was injected directly into the mammary glands of the mice in later gestational period through the papillary ducts to transfect the glandular cells in vivo. The milk of the mouse in lactation period was obtained and the tPA expressed in the milk was detected at a level of 80 ng/ml. Conclusion It is a simple and effective method for testing the expression ability of special vectors by transfecting the vectors directly into the mammary glandular cells in vivo.

目的 构建带HLA-B27基因的转基因大鼠,进一步研究HLA-B27 与强直性脊柱炎(AS)的发病原因及其机理.方法 应用转基因技术,把已克隆的人类AS基因HLA-B27基因组DNA导入Wistar大鼠受精卵中,并移植到假孕母鼠输卵管内,21 d后,出生27只原代大鼠.结果 经过PCR检测阳性后,再继续进行Southern印迹分析,确认3只大鼠为带HLA-B27基因转基因大鼠,整合率为11.11 %,其中1只转基因大鼠出现外周关节炎及脊柱强直表现.结论 本研究进一步表明HLA-B27 与AS的强相关性,为研究AS病因病理及治疗打下了基础.