Lei Ren1, Limei Zhang, Yanhong Liu. 1. Department of Laboratory Medicine, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, People's Republic of China.
Abstract
OBJECTIVE: To construct the eukaryotic expression vectors of mutant GPIIIa, establish CHO cell lines stably expressing mutant GPIIIa. METHODS: Total RNA were extracted from HEL cells. Mutant GPIIIa cDNA was synthesized by RT-PCR using the specific primers designed according to Genbank by Primer 5, then leaded to T1565C. The expression vector pcDNA3.1(+) and PCR products were respectively digested by NheI and HindIII, the specific cDNA fragments were directly inserted to the pcDNA3.1(+) because of having the same adhesive ends. Then wild type pcDNA3.1(+)IIIa and mutant pcDNA3.1(+)IIIa were respectively transfected into CHO cells using Lipofectamine 2000 reagent. The cell lines expressing GPIIIa and GPIIIa(T1565C) were screened by G418. Expression of GPIIIa and GPIIIa(T1565C) on transfected CHO cell surface were evaluated by flow cytometry and by RT-PCR to substantiate mRNA. RESULTS: The cDNAs of GPIIIa and GPIIIa(T1565C) were amplified by RT-PCR, and the recombinant of mutant pcDNA3.1(+)IIIa were constructed. By sequencing and enzyme digestion, it was be confirmed that there is a mutant of GPIIIa on 1565(T --> C). The result of flow cytometric analysis showed fluorescence intensity in the CHO cells transfected by recombinant is much higher than that by pcDNA3.1(+)IIIa. CONCLUSIONS: (1) Succeeded in constructing recombinants pcDNA3.1(+)IIIa(T1565C). (2) Succeeded in getting the cell lines expressing GPIIIa(T1565C).
OBJECTIVE: To construct the eukaryotic expression vectors of mutant GPIIIa, establish CHO cell lines stably expressing mutant GPIIIa. METHODS: Total RNA were extracted from HEL cells. Mutant GPIIIa cDNA was synthesized by RT-PCR using the specific primers designed according to Genbank by Primer 5, then leaded to T1565C. The expression vector pcDNA3.1(+) and PCR products were respectively digested by NheI and HindIII, the specific cDNA fragments were directly inserted to the pcDNA3.1(+) because of having the same adhesive ends. Then wild type pcDNA3.1(+)IIIa and mutant pcDNA3.1(+)IIIa were respectively transfected into CHO cells using Lipofectamine 2000 reagent. The cell lines expressing GPIIIa and GPIIIa(T1565C) were screened by G418. Expression of GPIIIa and GPIIIa(T1565C) on transfected CHO cell surface were evaluated by flow cytometry and by RT-PCR to substantiate mRNA. RESULTS: The cDNAs of GPIIIa and GPIIIa(T1565C) were amplified by RT-PCR, and the recombinant of mutant pcDNA3.1(+)IIIa were constructed. By sequencing and enzyme digestion, it was be confirmed that there is a mutant of GPIIIa on 1565(T --> C). The result of flow cytometric analysis showed fluorescence intensity in the CHO cells transfected by recombinant is much higher than that by pcDNA3.1(+)IIIa. CONCLUSIONS: (1) Succeeded in constructing recombinants pcDNA3.1(+)IIIa(T1565C). (2) Succeeded in getting the cell lines expressing GPIIIa(T1565C).
Authors: A D Michelson; M I Furman; P Goldschmidt-Clermont; M A Mascelli; C Hendrix; L Coleman; J Hamlington; M R Barnard; T Kickler; D J Christie; S Kundu; P F Bray Journal: Circulation Date: 2000-03-07 Impact factor: 29.690
Authors: E J Weiss; P F Bray; M Tayback; S P Schulman; T S Kickler; L C Becker; J L Weiss; G Gerstenblith; P J Goldschmidt-Clermont Journal: N Engl J Med Date: 1996-04-25 Impact factor: 91.245