Cell and Tissue Targeting
Cell and Tissue Targeting
Currently, the life cycle and biology of adenovirus (A d ) infected cells have been well characterized. In A d virus infection and transgene expression, there are three necessary sequential steps: (i) viral binding to cell surface receptors; (ii) the process of internalization; and (iii) transfer of the viral genome to the nucleus. The experimental approach in this chapter describes the construction scheme of A d viruses, i.e., how to utilize these viruses to target, bind and enter IE cells via - or secondary A d receptors (transduction process), and the expression of exogenous genes carried by these viruses in the target cells (transcription and translation) Author(s): T. Friedman et al.
Operation method
A d carrier for transduction-targeted construction of RG D-modified fibrils Move A d carrier for transduction-targeted construction of RG D-modified fibrils material Construction of shuttle plasmid vectors for cilia modification 1. Isolation of the ciliary region fragment of anti-Aminobenzylpenicillin from the recovered plasmid. a. EcoRI digestion of 10 tons of anti-Aminobenzylpenicillin PAdEasy-I plasmids for 4 h. b.1 % agarose gel recovery of 9. 6kb DNA fragment. c. Ligation cyclization. The resulting new plasmid (pFiberdE3-A m p ) has an intact ciliated region. 2 - Isolation of anti - kanamycin resistance gene. The gene was isolated by EcoRI digestion of IOfig pABS.4. a. EcoRI digestion of IOfig pABS. b- Enzymatic ends were complemented with K l e n o w fragments. c. Cyclize the recombinant plasmid (pA B S .4A E c o R I ) with ligase. d- Enzymatic cleavage of PA B S .4A E c o R I with EcoRI. e. Complementation of the B a w x H I site with Klenow fragments. f- Enzymatically cut PA B S .4A E c o R I with & out 1 . The combined I.3k b fragment contains the kana resistance gene and the two terminal SttaI sites. 3_Insert the canna-resistance gene into the anti - ampicillin cilium vector. a. Enzymatically digest pFiber-d E 3-A m p with M /eT [. b- Complementation of the M fe 1 [site with a K l e n o w fragment . c- Enzymatic cleavage of pFiber-d E 3-A m p with BsiBI . d- Insertion of the flat-ended fragment of I.3k b into the M / el-BsiBI site of pFiber-dE3-Amp . The synthesized vector (pFiber-dE3 ) includes the pili region and the ampicillin and canna resistance genes. Construction of the mutated cilia k n o b gene (Fig. 3) 4.P C R Preparation of complementary R G I > 4 C fragments. b- Add 30 umol/L of primer C (upstream primer) and primer D (downstream primer) to 500n g of pFiber-d E 3 to prepare (R G D --4C )-M/e I fragments. c-Set up the PCR as follows: 94°C denaturation l O m i n , 35 cycles (94°C , 30s, 50°C , 30s, 72°C , 60s), 72°C , IOmin d. Recover NAeI-(RGI> 4 C ) (1199bp) and (RG]>4C )-M/eI (180bp) with 2 % agarose gum. 5 . Preparation of mutant cilia k n o b sequences. a. Mix the two fragments M ieI-(R G I M C ) and (R G I >4C )-M / e I . b. Amplify a 500 ng mixture by PCR with no additional primers under the following conditions: 94°C (denaturation 4 min 30s, 10 cycles (94°C, 60s, 52°C, 60s, 72°C, 4 min), 72°C, 6 min. c. The PCR product (l352 bp) was recovered with 1 % agarose gel. 13. After I d of transfection, cells are inoculated into 96-well plates and screened with medium containing SOOug/ml G 418 until approximately 1/3 cell coverage. 14. Expand the cells appropriately for subsequent analysis. Purification of CAR/hEG F fusion protein 15. Collect medium from 293 cells stably expressing C A R /h E G F. 16. Add an equal volume of pre-cooled ammonium sulfate to precipitate proteins. 1 7 . Collect the precipitate by centrifugation and dissolve it in the original volume of 1 / 2 0 P B S . 18. dialysis with P B S. 19. purification of recombinant proteins with T A L O N cobalt-containing metal affinity chromatography resin (Clontech). 20. Dialyze with P B S. 21.Detection of expressed proteins by W e s t e r n blotting. 22. Detection of targeting efficiency of junction proteins and A d carriers. a. Bind the junction protein to the A d vector. b . Investigate the targeting efficiency of E G F R -tagged A d transfected cells expressing E G F R . reagents Agarose Strain: B J5183 and D H 5a Cell line: 293 cells Cesium chloride ethanol glycerin Canthaxanthin (25ug/m l ) For more product details, please visit Aladdin Scientific website.
a- Add 30 umol/L of primer A (upstream primer) and primer B (downstream primer) to 500 n g of pFiber-d E 3 to prepare withdraw eI-(R G I >4 C ). b- Add 30 umol/L of primer A (upstream primer) and primer B (downstream primer) to 500 n g of pFiber-d E 3 to prepare withdraw eI-(R G I >4 C ).
12. Transfect 293 cells with linear vector.
A d E a s y System (Qbiogene)
