Gene expression on Rat1 fibroblast cells after transformation by EVI1. Rev. Ang. de Ciênc. da Saúde. 2021 Jan – Jul 1 (1): 21-23

RESULTS The protein absorbance obtained from the cell lysates showed a moderate difference between Rat1neo(0.147ng/µl) and Rat15.6 (0.1665ng/µl) as revealed in the spectrophotometer and for Western blot analysis,both samples had to be adjusted to same concentration for 20µl.In the figure 1 the proteins analysed by Western blot are presented. This shows the expression of EVI1protein in Rat1neo and Rat15.6 with a migration band around 149 KDa.GAPDH protein showed the same thickness bands between Rat1neo and Rat15.6 samples, whereas, forCAIII the bands were thicker in Rat1neo and thinner for Rat15.6.The absorbance was made in duplicate, so a mean was calculated between the two values. The resultsfrom protein absorbance and RNA quantification are shown on Table 1.

Table 1- Protein Absorbance and RNA Quantification for Rat1neo and Rat15.6

Conc.

Rat15.6

Rat1neo

20µg BSA

10µg BSA

5µg BSA

2.5µg BSA

1.25µg BSA

 

0.625µg BSA

0µg BSA

RNA (ng/µl)

137.401

404.145

-

-

-

-

-

 

-

-

The RNA quality was checked by agarose gel electrophoresis and two bands of ribosomal RNA, 28S and18S, were observed which indicated the RNA quality obtained for this experiment.The qPCR results of CAIII gene expression in Rat1neo and Rat15.6 cells. The CAIII was highly expressedon Rat1neo whereas in Rat15.6 it was expressed at a reduced level because of the EVI1 gene overexpressingand the CAIII gene is repressed.After bacteria culture for transformation, there was no bacterial growth on some plates as indicated in Table2 and to complete the experiment, samples for Falcon tubes 1 and 2 were taken from classmates.

Table 2- Number of colo n ies count e d for bacteri al transformation

Plate

1

2

3

4

5

6

Plasmid

1

2

3

4

pUC19

Control

#colonies

0

0

118

141

16

0

The absence of bacteria growth for plasmid 1 and 2 may be related to the quantity of plasmid to give antibioticresistance to the DH5 α E. coli strain, coinciding with the negative control (no plasmid- no bacterial growth inpresence of ampicillin).Plasmid DNAs were amplified and identified in bacterial growth E. coli strain DH5 α and 1µl of Kpn1 enzyme(Fermentas-Life Science) was used to digest different plasmid DNAs in different fragments in 1% agarose gelelectrophoresis.

According to the DNA fragmentation and compared to the hyperladder, plasmid 1 corresponded to pBluescriptKSIIbecause it migrates at around 3100bp band, plasmid 2- pCAIIIUC with two fragments at 5500-1750bp; plasmid 3-pGL3 and plasmid 4- pRLCMV. The digest enzyme Kpn1 cut different plasmid DNAs in different fragments as

observed in agarose gel electrophoresis.Approximately 40 percent of pEGFP -N1 transfection was achieved in Rat15.6 fibroblast cells and about 1

percent for Rat1neo fibroblast cells.

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