A successive three-step 'Gap-repair' method to generate the mWAP-hLF hybrid gene locus / 生物工程学报

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin (hLF) genomic sequence is directed by the up & down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step 'Gap-repair' method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using 'Gap-repair 'method mediated by Red recombination system of lambda-prophage in Escherichia coli, in the first step, the 8 kb 3' flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5' flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5' & 3' flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.

Pharmacodynamics of ferrihemoglobin formation by 4-DMAP under hypoxic condition / 解放军医学杂志

Objective To guide the therapy for cyanide poisoning patients on plateau, the effect of hypoxia on the efficiency of 4-(N, N-dimethyl)-aminophenol (4-DMAP) in ferrihemoglobin (MHb) formation was investigated. Methods Rabbits were raised in hypoxic condition (4500m altitude) as acute-hypoxic animal model, and MHb concentration was determined by spectrophotography at the length of 635nm after 4-DMAP injection. Results There was no difference in hemoglobin content and MHb concentration between acute acutehypoxia group and controls. But in hypoxic rabbits, MHb formation as induced by 4-DMAP was improved distinctly. 30%-40% MHb concentration formation (optimal anti-cyanide concentration) was induced by 20mg/kg 4-DMAP in hypoxic rabbits, compared to 25-30mg/kg in control rabbits. However, when hypoxic rabbits received 25-30mg/kg 4-DMAP, which was the optimal dose for treatment of cyanide poisoning in normoxic animals, the animals manifested even worse symptoms of hypoxia, eren death occurred. Moreover, the level of MHb in acute-hypoxic models maintained longer than that in controls. The effect of 4-DMAP in MHb formation was closely related with duration of hypoxia. During hypoxia period from 1d to 5d, MHb concentration was increased gradually along with the duration of hypoxia, peaking on the 5d, and it started to decline on 7d, but it was still higher than that in control group. Conclusion Hypoxia enhances the effect of 4-DMAP in MHb formation and lowers the metabolism of MHb.