6.7K Views
•
08:54 min
•
March 29th, 2019
DOI :
March 29th, 2019
•0:04
Title
0:40
Overview of the REMOTE-control System
1:50
Modify the Gene of Interest for Repression by REMOTE-control
6:04
Manipulate Gene Expression In Vivo
6:50
Results: In vivo Repression and Activation Capability of the REMOTE-control System
8:23
Conclusion
Trascrizione
The significance of our method is its versatility and potency. It allows researchers to have robust control over endogenous gene expression in ways that have been challenging to achieve using existing methods. It allows researchers to investigate a gene's function at various expression levels and in a spatio-temporal manner.
Thus it allows for testing the reversibility of a phenotype, which is useful when studying disease related genes. To accomplish repression, the target gene intron is engineered to contain repron R, which contains 12 symmetric Lac operators. When the LacIGY repressor is expressed from the desired tissue specific promoter, the target gene is repressed.
Repression of the target gene can be reversed or adjusted to the desired expression level by administration of IPTG, an antagonist of the LacIGY repressor. To accomplish upregulation, the target gene promoter is engineered to contain four or more Tat operators, T, as binding sites for the rtTA-M2 activators. When the activator is expressed from the desired tissue-specific promoter in the presence of doxycycline, upregulation of the target gene is induced.
Upregulation of the target gene can be reversed or adjusted to the desired expression level by withdrawing or altering the concentration of doxycycline. To accomplish both repression and activation, the Lac repressor and Tat activator systems may be combined. To modify the gene of interest for repression, a transcriptionally inert intron towards the five prime end of the gene of interest should be identified for insertion of the repron sequence.
To obtain the genomic sequence for the gene of interest, navigate to the UCSC genome browser and select the latest draft of the mouse genome under the genomes tab. Enter the name or symbol of the gene of interest into the search bar to view the transcripts for the gene and then click go. Then, select the desired transcript variant for the gene of interest and click on the gene symbol next to the transcript variant of interest.
Next, under the sequence and links to tools and databases banner, click the genomic sequence link. For sequence retrieval region options, select only exons, introns and the default one FASTA record per gene. For sequencing formatting options, select exons in upper case, everything else in lower case and mask repeats to N.Then, click submit.
Finally, save this sequence, preserving the upper and lowercase formatting in a document or program that can be annotated. To avoid interruption of the CpG islands, select show for the CpG islands track under the expression and regulation banner of the UCSC genome browser and click refresh. Zooming in on the five prime introns, click on each CpG island shown in green, and select view DNA for this feature.
After selecting mask repeats to N, click get DNA to obtain the CpG islands sequence. Finally, overlay these sequences with the original sequence file, and annotate these as intronic regions to avoid. To avoid intronic regions with enhancer signatures in the tissues of interest, navigate to the ENCODE data base and select the experiments icon.
For the assay type, select ChiP-seq or DNase-seq and populate the other categories according to the cells to be engineered. After feature selection, select the left most pictogram in blue, for which view results as list appears. Then, select the data sets for the targets of h3k4 monomethylation, h3k27 acetylation, DNase 1, and CTCF that most closely matched the cells to be engineered.
Within each relevant data set, scroll to the file section, verify that the mm10 and UCSC are selected, and click the visualize button. Now, in the UCSC genome browser, zoom in on the five prime introns and click on each peak in the annotated peak tracks. Obtain the DNA sequence for each peak region by clicking on the chromosomal coordinates for each peak.
In the view drop-down menu, select DNA, and click mask repeats to N.Finally, overlay these sequences with the original sequence file and annotate these as intronic regions to avoid. To identify an sgRNA in the remaining intronic regions with high specificity and predicted efficiency scores, navigate to an online sgRNA design tool of choice, such as CRISPOR. Enter the sequence of the intronic region of interest, specify the relevant reference genome, and select the desired protospacer adjacent motif.
Then, click submit. Next, sort the predicted sgRNA's by specificity score and select one or more sgRNA's that also have a high predicted efficiency score. Finally, design a DNA template containing a PITT landing pad sequence flanked on both sides by 60-base homology arms that correspond to the sgRNA cut site.
For reversal of target gene repression, administer IPTG in the drinking water of the homozygous bred mice with the modified allele of interest by fully dissolving the desired amount of IPTG in sterile distilled water on the day of administration. Wrap the bottle with foil and administer the IPTG water in a light protected bottle to the mice of the appropriate genotype and controls for at least one week. Proceed to analyzing the expression of the gene of interest in the target tissue.
To induce the gene upregulation, administer doxycycline in the diet for a week and proceed to analyzing the expression of the gene of interest in the target tissue. qRT PCR anaylisis showed that DNMT1 expression was repressed to 15%of the unregulated levels using the promoter-based approach. The repression was reversed in a dose-dependent manner by treating mice with varying amounts of IPTG.
The observed DNMT1 repression and the reversal of DNMT1 repression by IPTG treatment was validated at the protein level by immunostaining. qRT PCR analysis of mKate2 expression showed that the intron-based approach achieved more than 90%repression from operators located several kilobases downstream of the transcription start site by attenuating transcription elongation. Confocal images of the mKate2 expression in the small intenstine of the mice with or without the LacIGY repressor validated the intron-based approach.
Robust upregulation and downregulation of DNMT1 expression were achieved in embryonic stem cells containing the modified endogenous DNMT1 allele with Tat and Lac operator sequences. Both regulations were fully reversible and inducible by IPTG and Dox treatments. Strong upregulation of DNMT1 was observed from the liver, spleen, and kidney.
However, no detectable upregulation in the heart was observed, suggesting that the cell cycle-dependent expression pattern of DMNT1 and the scarcity of proliferative cells in the heart may underlie this observation. Studying the in vivo function of a critical gene by manipulating its expression has often been challenging due to lethality. Our method allowed us to overcome the lethal phenotype for our gene of interest and enabled us to study its role in tumorogenesis in vivo.
Likewise, this technology will enable investigation of other essential genes that have been difficult to study.
This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.