Effect of cryopreservation on biological activity of national standard of human granulocyte colony-stimulating factor after reconstitution / 中国生物制品学杂志

@#ObjectiveTo investigate the effect of cryopreservation on the biological activity of the national standard of human granulocyte colony-stimulating factor(hG-CSF)after reconstitution,so as to provide a reference for the use of the national standard of hG-CSF.MethodsThe biological activity of the standard was determined according to the general rule 3525 of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition);The reconstituted hG-CSF national standard was aliquoted and stored at-80 ℃,-40 ℃ and-20 ℃,and then sampled at 1,2,3,5 and 6 months to detect the biological activity. The standards reconstituted before use were used to quantify the standards stored at -80 ℃ for different time durations,and the standards stored at -80 ℃ were defined as the reference of 100% activity to quantify the relative biological activity of the other samples.ResultsThe Eyrlng equation fitted by the thermal acceleration stability experiment was:ln {k(t)} = 6. 75-3 772. 20/T + ln(T),R~2= 0. 969. The biological activity of hG-CSF national standard was predicted to decrease by 5% after about 93. 4 months storage at -80 ℃；The biological activity of reconstituted standards frozen at -80 ℃ decreased by about 24%.ConclusionThe aliquoted reconstituted hG-CSF national standard can be stored at -80 ℃ stably for more than half a year. However,freezing and thawing will cause the activity value to drop by more than 20%,so it is not recommended to reuse after reconstitution.

Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Ultrasonographic evaluation of hG-CSF transgenic goat conceptus / Avaliação ultrassonográfica de conceptos transgênicos para o hG-CSF

The objective of this study was to evaluate the development of transgenic (T) goat embryos and fetuses for human Granulocyte Colony Stimulating Factor (hG-CSF) by ultrasonography. Four pregnancies in non-transgenic (NT) goats were obtained after fertilization (either fixed-time artificial insemination or natural mating) using the T male for hG-CSF. Ultrasound examinations were carried out at 30, 40 (transrectal via), 50, 60, 90 and 120 days of pregnancy (transabdominal via). Some parameters were observed such as morphology, organogenesis and formation of skeletal fetuses, viability with cardiac activity and fetuses movements. Measurements were taken of the crown-rump length, diameter of embryonic vesicle, thorax, abdomen, umbilical cord and placentomes. After parturition, DNA testing was conducted in all offspring and 4 T and 2 NT kids were identified. The conceptus started their differentiation at 40 days. The heart was detected in all examinations and the heart chambers were assessed at 50 days. Gastric compartments, liver and kidneys were observed at 60 days, the same period that all bony structures were visualized. Average values of all evaluated parameters had a gradual increase with the progression of pregnancy. T and NT goat embryos and fetuses had a similar growth and all remained viable throughout the experimental period.

O objetivo deste estudo foi avaliar o desenvolvimento de embriões e fetos transgênicos (T) para o Fator Estimulante de Colôniasde Granulócitos humano (hG-CSF) por ultrassonografia. Quatro gestações em cabras não transgênicas (NT) foram obtidas pos fecundação (inseminação artificial em tempo fixo ou monta controlada) utilizando o bode T para o hG-CSF. Exames ultrassonográficos foram realizados aos 30, 40 (via transretal), 50, 60, 90 e 120 dias de gestação (via transabdominal). Alguns parâmetros foram observados como morfologia, organogênese e formação do esqueleto fetal, viabilidade por meio de atividade cardíaca e movimento fetal. As seguintes mensurações foram realizadas: comprimento crânio caudal, diâmetro da vesícula embrionária, do tórax, do abdomen, do cordão umbilical e dos placentomas. Após o parto, o exame por PCR foi conduzido em todas as crias e 4 T e 2NT foram identificadas. O concepto iniciou sua diferenciação aos 40 dias. O coração foi detectado em todos os exames e as câmeras cardíacas foram identificadas aos 50 dias. Compartimentos gástricos, fígado e rins foram observados aos 60 dias, o mesmo período que todas as estruturas ósseas foram visualizadas. Valores médios de todos os parâmetros avaliados tiveram um aumento gradual com o avanço da gestação. Embriões e fetos T e NT tiveram um crescimento similar e todos permaneceram viáveis durante o período experimental.

Production of transgenic goat (Capra hircus) with human Granulocyte Colony Stimulating Factor (hG-CSF) gene in Brazil

In order to produce transgenic goats with hG-CSF, a total of 24 adult Saanen and 48 adult undefined breed goats were used as donors and recipients, respectively. Donors were estrus-synchronized with vaginal sponges and superovulated by a treatment with 200 mg FSH given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced by injecting 100µg GnRH 36 h after sponge removal. The recipients also received an estrus synchronization treatment. Donors were mated with fertile Saanen bucks and, approximately 72 h after sponge removal, zygotes were recovered surgically by flushing oviducts. The recovered zygotes were briefly centrifuged to a reliable visualization of the pronuclei. The DNA construct containing hG-CSF gene flanked by goat and bovine alphas1-casein sequences was injected into pronuclei of 129 zygotes. The microinjected embryos (3-6 per female) were transferred to 27 recipients. Ten recipients became pregnant and 12 kids were born. One transgenic male founder was identified in the group of kids. This is the first report of a birth of a transgenic goat in Latin America.

A fim de produzir caprinos transgênicos para o hG-CSF, utilizou-se 24 cabras Saanen adultas e 48 cabras sem raça definida adultas como doadoras e receptoras, respectivamente. As doadoras tiveram o estro sincronizado por esponjas vaginais e foram superovuladas com 200 mg de FSH em doses decrescentes, duas vezes ao dia e iniciando 48 h antes da retirada da esponja. A ovulação foi induzida pela injeção de 100 µg de GnRH às 36 h após a retirada da esponja. As receptoras também receberam um tratamento de sincronização do estro. As doadoras foram cobertas por bodes Saanen férteis e, aproximadamente 72 h após a retirada da esponja, os zigotos foram colhidos cirurgicamente por lavagem dos ovidutos. Os zigotos colhidos foram rapidamente centrifugados para uma melhor visualização dos pró-núcleos. A construção de DNA, contendo o gene do hG-CSF flanqueado pelos genes caprino e bovino da alfas1-caseína, foi injetada em 129 embriões. Os embriões microinjetados (3 a 6 por receptora) foram transferidos para 27 receptoras que responderam ao tratamento. Dez receptoras ficaram gestantes e 12 crias foram produzidas. Um macho transgênico fundador foi identificado no grupo de crias nascidas. Este é o primeiro relato do nascimento de um caprino transgênico na América Latina.