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Transient transfection analysis was initally employed to assay the fimction o f p47^^®^CAT and control constructs in myeloid and non-myeloid cell lines. Transiently transfected cells were lysed and assayed after 18-72hrs. (section 2.11.3). The efficiency o f transfection was quantitatively determined by spectrophotometric assay o f B- galactosidase activity in cells co-transfected with the pSV-B-Galactosidase Control Vector (Promega).

250V

275V

300V

Figure 4.1 Optimisation of transfection of PLB-985 myeloid cells by electroporation. PLB-985 cells

were transfected by electroporation at constant capacitance (950|iF) and 250, 275 or 300V, as indicated. Transfection efficiency was assessed by in situ detection o f 6-Galactosidase expression directed by the pSV-B-Galactosidase Control V ector (Promega), after 24-72hrs.. Positive cells exhibit dark blue nuclear staining. Transfection efficiency ranged from approx. 2-8%, and was optimal at 275V.

Preliminary experiments were assayed by detection o f CAT protein levels using an ELISA kit (Boehringer Mannheim). A CAT-based antibody sandwich is used to detect CAT protein in cellular lysate. The final component, a peroxidase conjugate, catalyses a colour change reaction that can be quantified spectrophotometrically (section 2.2.16.3). Absolute levels o f CAT protein are determined by comparison to standard solutions o f CAT protein (Figure 4.2). Results determined by CAT-ELISA were generally o f a low level, and inconsistent for samples measured in triplicate. The results o f transfection experiments performed in the absence o f transfection efficiency correction are shovm in

Figure 4.3; results o f experiments performed by co-transfection with the pSV-B- Galactosidase Control vector are shown in Figure 4.4. Levels o f CAT protein detected in cells transiently transfected with CAT reporter constructs did not differ significantly from levels detected in untransfected cells (Fig. 4.3). Furthermore, no significant difference was determined between positive and negative control constructs, or between control and p47^^®^CAT reporter constructs (Fig. 4.3, Fig. 4.4). No conclusions concerning p47^^®^ promoter function could thus be drawn from these experiments.

Assay o f enzymatic activity is generally more sensitive than assays based on antibody detection. CAT enzymatic activity in cellular lysate is determined by the ability to acetylate a [*"^C]-labelled chloramphenicol substrate at either or both o f two hydroxyl groups. A protocol for assay o f CAT enzymatic activity by thin layer chromatography (TLC) was adopted since it provides a visual accoimt o f CAT activity. Differentially- acetylated reaction products are separated from residual substrate on a silica plate in the presence of chloroform and methanol, and visualised by autoradiography (section 2.2.16.4). Determination o f CAT enzyme activity in PLB-985 myeloid cells transiently transfected with p47^^^^CAT constructs is shown in Figure 4.5. Whilst absolute consistency of CAT activity was not observed between triplicate samples, a general trend o f promoter function for each construct is apparent. As expected, the negative control vector pCAT®-Basic directed levels o f CAT activity comparable to imtransfected cells. p47^^°^ constructs pCATXR and pCATXRS directed activity comparable to pCAT®-Basic, whilst pCATXRN-pCATXRl 2 directed significant levels o f CAT activity by comparison to pCAT®-Basic. Although use o f the TLC-based assay facilitated a broad determination o f p47^^°^ promoter function, transient transfection

2.5 T- <

I

§ u ë I

I

^ 0.5 - 0 0.125 0.25 0.5 1 CAT protein (ng/ ml)

Figure 4.2 Assay of CAT protein standards by CAT-ELISA. Absorbance at 405nm was determined by CAT-ELISA for CAT protein standards ranging from O-l.Ong/ml (Boehringher Mannlieim). Absorbance was measured in absorbance units (AU). Response was linear to approximately 0.5ng/ml.

2.5 T

El HL-60

m

PLB-985 □ AM12

CMV Basic pCATXR pCATXRS

CAT reporter constructs

Figure 4.3 Assay of CAT protein in cell lines transiently transfected with p47^*"^CAT and control CAT reporter constructs. HL-60, PLB-985 and AM 12 cells were transiently transfected with pAlCMVlXCAT.3 (CMV) and pCATO-Basic (Basic) control plasmids, and p47^*^CAT reporter plasmids as indicated. CAT protein levels were detected by CAT-ELISA. Absorbance readings were corrected for soluble protein content and expressed relative to the level of CAT protein detected in untransfected (wt) cells ± standard error (se).

m

PLB-985 ■ U937

CMV Basic pCATXRN pCATXRF

CAT re p o rte r constructs

Figure 4.4 Assay of CAT protein in cell lines transiently transfected with p47^*"'"CAT constructs and pSV-B-Galactosidase. PLB-985 and U937 cells were transiently transfected with pAICM VIXCAT.3

(CM V) and pCA T® -Basic (Basic) control plasmids and p47^^"'CAT reporter plasmids, as indicated. Co­ transfection was perform ed with the pSV-B-Galactosidase vector. CAT protein levels were detected by CAT-ELISA. A bsorbance readings were corrected for transfection efficiency and expressed relative to the

level o f CAT protein detected in pCA T® -Basic transfectants in each cell type (±se).

C o n s tr u c t S u b s tr a te in te n s ity / P S L P r o d u c t in te n s ity / PS L T o ta l in te n s ity / PS L % c o n v e r s io n M ean S ta n d a rd d e v ia tio n ( s . d . ) pCA TX R-1 97731.5 501.8 98233.3 0.511 -2 97424.4 510.9 97935.3 0.522 0.515 0.006 -3 93310.9 479.7 93790.6 0.511 pCATXRN-1 86247.5 581.3 86828.8 0.674 -2 81902.7 668.1 82570.8 0.809 0.713 0.084 -3 73771.4 487.3 74258.7 0.656 PCATXR12-1 51947.1 762.5 52709.6 1.447 -2 66297.7 895.8 67193.5 1.333 1.379 0.060 -3 63527.5 873.9 64401.4 1.357 PCAT269-1 60660.2 1193.1 61853.3 1.929 -2 54707.3 1054.0 55761.3 1.89 1.728 0.315 -3 54447.3 753.5 55200.8 1.365 pCATBasic-1 84095.3 467.4 84562.7 0.553 -2 62986.3 402.1 63388.4 0.634 0.604 0.045 -3 75179.9 473.5 75653.4 0.626 CAT 12310.6 16742.0 29052.6 57.627 57.627 - PLB985 wt 87185.8 623.1 87808.9 0.710 0.710 -

B

2.5 -■ 6 0 1 5 0 - 4Q- 3 0 - 20- 10- X R X R N X R 12 269 Basic PLB C A T XR XRN XR12 269 Basic PLB C A T re p o rte r c o n stru c t

F ig u re 4.5 (am m ended): P h o sp h o lm a g e an a ly sis of T L C data p re sen ted in F ig u re 4 .5 . TLC plates w ere exposed to a phosphoim age screen for 65 hours and analysed by the A ida V ersion 2.0 2D densitom etry program m e. C A T reporter gene activity in transiently transfected PLB 985 m yeloid cells assayed by T L C (Fig. 4.5) w as m easured in term s o f % conversion from unacetylated chloram phenicol substrate to acetylated reaction products. Substrate and product intensities w ere calculated by m easurem ent o f pixels p er defined unit area (PSL). A , T able o f data analysis for PLB 985 cells transiently transfected w ith p47P^^-^CAT reporter gene constructs (in tripli(^ate). B, G raph o f m ean % conversion for P L B 985 cells transiently transfected w ith p47P^^-^CAT reporter gene.C onstructs (XR, pC A T X R ; X R N , p C A T X R N ; X R 12, pC A T X R 12; 269, pC A T 2 6 9 ; B asic, pCATBasic; PLB,.i P L B 985 w t) +/-" s.d.. C, G raph o f m ean % conversion for PLB 985 cells transiently transfected w ith

CAT XR XRN XRP XR12^

_{1 r}

Basic 1 w t

f

F igure 4.5 Assay o f C A T activ ity in PLB -985 cells tra n sie n tly tra n sfe c te d w ith p47'’*"^CAT r e p o rte r c o n stru c ts. PLB-985 cells were transiently transfected with CAT reporter plasmids. Transfections were perform ed in triplicate. CAT activity o f ~80pg cellular lysate (as indicated) or 0.5U CAT protein (CAT) was determ ined by TLC. Reaction products are as follows: S, unacetylated substrate; M l, M2, mono-acetylated product; D, di-acetylated product. CAT activity determ ined for p47^^°^ constructs pCA TX R (XR), pCATXRN (XRN), pCA TX RP (XRP), pCA TX R 12 (XR12) and pCAT®-Basic (Basic) is shown in order to indicate the broad trend o f prom oter activity. CAT activity directed by the rem aining p47^*“ constructs was as described in the text. CAT activity determined in untransfected PLB985 cell lysate is shown for com parison (wt).

experiments were generally hampered by inefficient transfection efficiencies and the low survival rate o f electroporated cells (approx. 95% as indicated by trypan blue exclusion (data not shown)).

In document Paraísos fiscales, erosión tributaria y reformas fiscales en México (1997 2015) (página 183-187)