BIO 313 Zebra Fish Embryo.docx

This lane was negative for expression the next set of

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contained only the first set of primers and no RNA. This lane was negative for expression. The next set of wells corresponded to the second primer set for pax2b. Lane seven contained tissue 27hpf plus our second set of primers and had no expression. Lane eight also contained the second set of primers, but 75hpf. This lane showed multiple bands from 250-500bp. The last experimental well contained no RNA with primer set two. This lane was negative for expression. Lanes three, six, and nine acted as negative controls that tested for DNA contamination. Wells one, two, four, five, and eight show multiple bands on the gel (Fig.1). The gel electrophoresis of the pax1a/1b showed no expression. The expression seen was in well one with 27 hpf RNA plus -actin primers and this was a positive control. Lanes two through nine showed no expression. Discussion We know that if you do not span an intron you will not be able to tell id there is gDNA contaminate because the products will be the same size. Our results show that we successfully spanned an intron between two exons with our primers. This was important because we were 6
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able to tell if we had any contamination and could proceed to interpret our results from the gel electrophoresis. When a gene is expressed, the DNA of the gene is transcribed into pre-mRNA. The results show that genes can be regulated at different time points in development. By placing - Actin primers in with the RNA we were creating a positive control because we know that -Actin protein is produced at all time points in development. Pax2a was expressed at both 27 and 75 hours after fertilization. This suggests that the gene is needed at these points in development. The Pax2a had a very strong expression which means that it may be needed in many types of cells in a systematic fashion. Figure 1 shows that there are multiple bands in the gel. This provides some evidence on the alternative splicing of the DNA into different proteins. Pax2b had lighter expression and multiple bands in the gel. This lighter expression in pax2b suggests that the gene is may only be expressed in specific cells. Pax1a and pax1b did not show any expression (Fig. 1). The only result was in the positive control well. Because only one control was positive, it is hard not to suggest that the results are due to error. It is possible that the pax1 gene is not expressed in neither 27hpf nor 75 hpf, but because of the lack of control results we can not rule out the possibility of error in the procedure. We know that during development, cells start in a pluripotent state, from which they can differentiate into many cell types, and progressively develop a narrower potential. Gene-expression programs become more defined and locked in as the development enters its later stages (Wolf 2007). Our results fit into this knowledge of gene regulation because the expression of our genes were expressed at different times. With these results we can suggest that the stage in development plays a role in when certain genes are expressed, but we can not tell why. The duplicated B copy of pax2 shows that there is change in the expression from its 2a parent. The duplicated gene could be developing a new function. Futures experiments that could be done is look further into how these genes are being restricted and by which biological pathway. Knowing that the gene is expressed you can 7
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