Mediated Transcription Program - Essay Example

Results Three mice are used for the Early growth Response – 2 Mediated transcription program. They are Wild Type Mice (WT), EGR 2 –transgenic Mice and EGR2 Knock out Mice. The Knock out mice and the spleen was used for Micro array analysis technique, Thymus for RT-PCR analysis and spleen for FACS analysis. Thymus is the region where the T-cell maturation occurs. The matured T-cells are in turn responsible for the Auto immunity. The T-cell has receptors. The T-cell receptors undergo genetic rearrangement during maturation, enabling the cells to bear a unique T-cell receptor. Each T-cell receptor has a specific unique peptide: Major Histocompatibility Complex with them. The Spleen contains lymphocytes and another type of white blood cells called Macrophages. Thus spleen is also the best source of lymphocytes. The T-cells are usually divided into two major Subsets- T Helper cells and T-Killer cells. The T-helper cells are called as CD4+ cells and T-killer cells are called as CD8+ cells. Early growth response gene – 2, a zinc finger transcription factor is expressed in the CD44 cells. It also controls their activation and proliferation. In the absence of this EGR gene -2 at CD44high + cells, and not CD44low + cells, the T cells were found to be hyper reactive and hyperproliferative in vivo. (Zhu et. al, 2008) In this experiment, two types of cells were used for FACS research, Wild type and EGR-2 Transgenic mice. The cells extracted from the spleen are separated for the lymphocytes and then mixed with 1 μl of the antibodies. The antibodies used are CD3 P (Phycoerythrin)+CD4 F(Fluorescein), CD3 P+CD8 F, CD4 F+CD8 P. (labtestsonline.org, 2009). The number of the positive cells were calculated for the two types and compared with each other. Result 1: STAINING WITH CD4 (FITC SL) + CD8 (PE SL) The wild type and the EGR-transgenic mice spleenocytes were stained with CD4 (FITC SL) + CD8 (PE SL). The results have shown that the wild type mice has the normal count. The CD4 positive cells are greater than the CD8 cells normally. In transgenic mice, the CD4 and CD8 cells counts are nearly the same. The EGR-Transgenic mice are found to have greater CD8 cells than the wild type. Type CD8(PE) CD4(FITC) WT 5.51 41.96 Egr2 25.71 23.29 Result 2: STAINING WITH CD3 (PE SL) + CD4 (FITC SL) The wild type and the EGR-transgenic mice spleenocytes were stained with CD3 (PE SL) + CD4 (FITC SL). The results indicate that There was no characteristic difference in the CD3 positive cells in both wild type and EGR2. Whereas the CD4 positive cell count was nearly double in the wild type when compared to the EGR2 Tr. Type CD3 (PE) CD4(FITC) WT 11.18+24.75 24.75 Egr2 11.92+12.98 12.98 Result 3: STAINING WITH CD4 FITC +CD3 PE Type CD3 PE CD4 FITC KO 31+41 6+41 Tr 24+24 12+24 WT 13+18 2+18 The staining of all the three type spleenocytes with CD4 FITC +CD3 PE, has showed that the CD3 positive cells are found to be greater in number than the CD4 positive cells. The CD3 and CD4 cells are more in knock out type and transgenic type than wild type. Result 4: STAINING WITH CD4 FICT + CD8 Type CD8 PE CD4 FITC KO 41+5 13+5 Tr 50+6 9+6 WT 10+10 21+10 The staining of the spleenocyte cells with CD8 PE and CD4 FITC isolated from the wild type, knock out mice and transgenic mice shows that CD4 positive cells are found in greater number than the CD8 cells. The CD8 and CD4 cells are found to be more in knock out type and transgenic type than in wild type. RESULT 5: STAINING WITH CD3PE +CD8FITC The staining of the spleenocyte cells with CD3 PE and CD8 FITC isolated from the wild type, knock out mice and transgenic mice shows that CD3 positive cells are found in greater number than the CD8 cells. The CD3 and CD8 cells are found to be more in knock out type and transgenic type than in wild type. THYMUS: RESULT6: STAINING WITH CD4PE + CD8FITC: TYPE CD4PE CD8FITC KO 82+3 3+3 Tr 23+8 24+8 WT 79+5 4+5 The staining of the thymus cells with CD4 PE and CD8 FITC isolated from the wild type, knock out mice and transgenic mice shows that CD4 positive cells are found in greater number than the CD8 cells. The CD4 and CD8 cells are found to be more in knock out type and transgenic type than in wild type. RT PCR : The above diagram shows the RTPCR result of thymus of three types of mice using different primers. The above diagram shows the RTPCR result of the cDNA synthesize from the thymus. The primer represented by red colour shows significant difference in mice. Comparing the results of the three types a thick band for the Egr2 gene is got in case of the transgenic mice mice type. SPLEEN AND THYMUS: SPLEEN: Egr2 cKO Egr2 WT Egr2 Tr The above figure shows the six week old spleen size of Egr2 cKO mice, Egr2 WT and Egr2 Transgenic mice. By comparing the sizes of spleen of three types of mice the spleen size is greater for the knock out type when compared to the other two types. If the spleen size is bigger thean normal then it may lead to lymphoproliferative disorder which may lead to an increase in the production of lymphocytes in the body.(nlm.nih.gov, 2009). But the spleen size of the Transgenic mice type is smaller than that of the Wild type mice. THYMUS: Egr2 cKO Egr2 WT Egr2 Tr The above figure shows the six week old thymus size of Egr2 cKO mice, Egr2WT, Egr2 Transgenic mice. Thymus helps in the maturation the T lymphocytes and plays role in immunity. By comparing the size of thymus of the three types of mice types the size of thymus of the knock out type mice is found to be small whan compared to the transgenic and the wild type mice type. CONCLUSION: The Early growth response gene- 2 has the main function of coding the early growth response protein. The early growth response protein binds with the DNA and it helps in controlling particular genes. In this experiment the wild type, knock out and transgenic types of mice are analysed. The FACS research of the spleen and the thymus cells stained with different stains show that the transgenic type has a higher number of T cells when compared to the wild type. When stained with CD4 (FITC SL) + CD8 (PE SL), the number of cells is found to be greater in transgenic than in wild type. When the spleenocytes are stained with CD3PE +CD8FITC, CD4 FICT + CD8, CD3 (PE SL) + CD4 (FITC SL), the amount of CD4 and CD8 cells are found to be higher and the total amount of cells are found to be higher in transgenic type, out of the three. (Harris et. al, 2004) While comparing the spleen size, the spleen size in the Transgenic mice is found to be small. If the size of the spleen is small then the splenomegaly disorders may be avoided. (Zhang and Zhang, 2001). Thus it is concluded that Egr2 gene has a major function in the regulation of the immune system and the transgenic mice has a higher immunity when compared to the wild type mice. REFERENCE: Harris, J et. al. (2004).Early Growth Response Gene-2, a Zinc-Finger Transcription Factor, Is Required for Full Induction of Clonal Anergy in CD4+ T Cells1. The Journal of Immunology. 173: 7331-7338. Retreived on September 29, 2009 from http://www.jimmunol.org/cgi/content/abstract/jimmunol%3B173/12/7331 labtestsonline.org. (2009). CD4 and CD8. Retreived on September 29, 2009 from http://www.labtestsonline.org/understanding/analytes/cd4/test.html nlm.nlh.gov. (2009). Spleen Disease. Retreived on September 29, 2009 from http://www.nlm.nih.gov/medlineplus/spleendiseases.html Zhang, Z X and Zhang, L. (2004). CD3+CD4-CD8- aß-TCR+ T cell as immune regulatory cell. Journal of Molecular Medicine. 79( 8), 420. Retreived on September 29, 2009 from http://www.springerlink.com/content/1lp0rt4da7636r3j/ Zhu, Bo et. al. (2008). Early growth response gene 2 (Egr-2) controls the self-tolerance of T cells and prevents the development of lupuslike autoimmune disease. Journal Experimental Medicine. The Rock feller University Press. 205(10). 2295–2307. Retreived on September 29, 2009 from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2556781 Read More