L1 Insertion Element Influence The GFP Expression

Objective: The completion of genomic sequencing has made it apparent that mammalian genomes are littered with enormous numbers of transposable elements interspersed within and between single-copy endogenous genes. LINE-1 or L1(long interspersed nuclear elements 1) is the most representative in these transposable elements. L1 elements are the most abundant autonomous retrotransposons in the human genome, accounting for about 17% of human DNA. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 has two open reading frames, encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. So far we have discovered that L1 is concerned with many biological phenomenas, such as gene mutation, X-chromosome inactivation, genome rearrangement, gene expression silencing, tumor formation, organic evolution and so on. In the immune system, allelic exclusion about TCR and BCR is related to L1. The gene expression of IL-2 and IL-4 is also associated with L1.Recently, research has shown that L1 has the potential to down-regulation the gene expression. Han fused either LacZ or L1.2 ORF2 coding regions downstream of the green fluorescent protein(GFP). The presence of ORF2 sequence significantly lowered steady-state protein production and led to a 70-fold decrease in RNA relative to LacZ. When the reading frame of ORF2 has changed, the expression of GFP was still inhibited. Han also observed that inserting ORF2 in the antisense orientation produced a similar, but less potent, decrease of the GFP RNA and protein expression.L1 includes a great of subfamilies, there are some differences in the sequences. It is yet unkown that the same result in another subfamily as in L1.2 exists or not. In addition, some experiments showed that in the L1-EGFP transgenic mice, green fluorescence was observed in the testes of mice instead of another organism. It is obviously that L1 plays different role among different cells. So it is quite necessary to do some researches about the factors that L1 regulates the gene expression.For this reason, we chose the ORF2 as the object of this research. ORF2 comes from L1PA3. Through observing the gene expression regulated by ORF2, we aimed at making clear the factors that L1 regulates the gene expression. That will supply the experimental evidence to explore the underlying mechanism of L1 regulating the gene expression.Methods: 1 The primers of the targeting sequences were designed according to the principle of primer designation. The targeting sequences amplified by PCR were used for insertion elements. According to the experiment, suitable enzyme digestion spots were introduced into the primers.2 We selected pEGFP-C1 as the vector. The vector and insertion element were digested with the same restriction enzymes. The ligation reaction was conducted with T4 DNA ligase. The recombinant plasmid was transformed into strain DH5α. The positive clone was selected by PCR. The plasmid identified by restriction enzyme was used for sequencing.3 The recombinant plasmid was transiently transfected into HeLa cells by LipofectamineTM2000. GFP expression in HeLa cells was observed by fluorescence microscope. The total cell numbers was counted (at least 500 cells) with the light microscopy. The GFP positive cell numbers was counted with the fluorescence microscopy in the same scope. Take pictures respectively.Results:1 Identify the recombinant plasmid p-ORF2 and p-ORF2as were identified by restriction endonuclease (XhoⅠand PstⅠ) cleavage and sequencing. The recombinant plasmids were successfully constructed. p-LacZ and p-LacZas were identified by restriction endonuclease (XhoⅠand PstⅠ) cleavage and sequencing. The recombinant plasmids were successfully constructed. p-ORF2as/A, p-ORF2as/Am and p-Aas-ORF2as /Am were identified by sequencing. The recombinant plasmids were successfully constructed. 2 The influence of GFP expression by inserting ORF2, ORF2as,LacZ and LacZasBy inserting ORF2,ORF2as,LacZ and LacZas downstream of GFP, we constructed p-ORF2,p-ORF2as,p-LacZ and p-LacZas. Using pEGFP-C1 as the control, these five plasmids were transiently transfected into HeLa cells. The GFP expression percent are: p-ORF2(0.53±0.10)% p-ORF2as(2.28±0.66)% p-LacZ(1.82±0.31)% p-LacZas(1.07±0.33)% pEGFP-C1(23.88±0.93)%3 The influence of GFP expression by inserting ORF2as/A, ORF2as/Am and polyas-ORF2as/AmWe chose ORF2as as the object of this research. First,we altered the enzyme digestion spot of ORF2as and constructed p-ORF2as/A. Second, we altered the reading frame of ORF2as/A and constructed p-ORF2as/Am. Finally,we inserted polyAas upstream of ORF2as/Am and constructed p-Aas-ORF2as/Am. Together with p-ORF2as and pEGFP-C1, these five plasmids were transiently transfected into HeLa cells respectively. The GFP expression percent are: p-ORF2as(2.5±0.92)% p-ORF2as/A(10.6±0.65)% p-ORF2as/Am(0.39±0.10)% p-Aas-ORF2as/Am(12.53±0.87)% pEGFP-C1(24.13±1.43)%4 Analysis the GFP expression(1) The GFP expression percent of p-ORF2, p-ORF2as, p-LacZ and p-LacZas were significantly lower than pEGFP-C1(P<0.01).(2) The GFP expression percent of p-ORF2 was lower than p-ORF2as, p-LacZ and p-LacZas(P<0.05).(3) The GFP expression percent of p-ORF2as/A was higher than p-ORF2as(P<0.01).(4) The GFP expression percent of p-ORF2as/Am was significantly lower than p-ORF2as/A(P<0.01).(5) The GFP expression percent of p-Aas-ORF2as/Am was significantly higher than p-ORF2as/Am(P<0.01).Conclusion:1 The downstream of GFP was inserted with targeting sequences in the pEGFP-C1. The size of the insertion elements is same, but the sequences are different. We found all the insertion elements can inhibit the GEP expression. The inhibition of ORF2 is the strongest.2 When the same ORF2as sequences inserted downstream of GFP at different enzyme digestion spot, the inhibition to GFP expression was different.3 Reading frame change of ORF2as inserted downstream of GFP at the same enzyme digestion spot influenced the GFP expression.4 When SV40 polyA was inserted in the antisense orientation between GFP and ORF2as/Am, it could decrease the inhibition by ORF2as/Am.