The overall goal of this procedure is to map integration sites of murine leukemia virus derived retroviral vectors to define cell specific regulatory regions. This method can help answer key question in gene therapy and gene regulation studies such as where the vector integrates in the human genome and which regulatory element can actively use via particular sub type. We first had the idea for this method observing that in gene therapy studies, MLV vectors integrate into active sub specific promoters and enhancers.
Begin by transducing the target cells with an MLV-derived retroviral vector harboring the EGFP reporter gene and pseudotyped with VSV-G or the amphrotopic envelope glycoprotein. Mock transduced cells are used as a negative control. 48 hours after transduction, centrifuge the samples and then resuspend 100, 000 cells in 300 microliters of PBS containing two percent fetal bovine serum.
Then, measurement the EGFP expression by flow site metric analysis. Harvest another 0.5 to five million transduced cells for genomic DNA preparation and subsequent restriction enzyme digestion. Set up four restriction enzyme digestions per sample in 1.5 milliliter tubes.
Digest 0.1 to one microgram G-DNA in each tube. By adding 10 units of TRU91 and one microliter of buffer M per sample in a final volume of 10 microliters. Dispense two microliters of the mix into digestion tubes.
And then incubate the reactions at 65 degrees celsius for six hours or overnight. Following the incubation, add 40 microliters of water to a 1.5 milliliter tube. In the same tube, add 10 units of PST1 and one microliters of buffer H per sample.
Dispense 10 microliters of the digestion reaction mix in the digestion tubes. Incubate the reactions at 37 degrees celsius for six hours or overnight. The next day, set up eight linker ligation reactions in 1.5 milliliter tubes.
For each reaction, add the ligase reaction mixture to 10 microliters of the digestion genomic DNA. Incubate at 16 degrees celsius for three to six hours. For first-round PCR, set up 48 PCR reactions in 0.2 milliliter tubes.
For each PCR, add the reaction mixture containing the linker primer and MLV3 prime LTR primer to two microliters of the ligation reaction. Place the samples in a thermal cycler with heater lid. Set the cycling parameters and run the first round PCR reaction.
For the second round PCR, set up the reaction mixture including linker nested primer and MLV3 prime LTR nested primer. Then, add two microliters of the PCR product from the first-round PCR as template. Then, run the second round PCR reaction to generate the final LN PCR products.
After the second round PCR, pull the 48 nested LM PCR reactions in a 15 milliliter tube. Then add 20 microliters of loading buffer to 100 microliters of the pooled and precipitated LM PCR product. And load the sample onto a one percent agarose gel together with the 100 base pair DNA ladder.
Run the gel at five volt per centimeter for 30 to 60 minutes once the bands are resolved, cut the proportion of the gel containing 500 to 150 base pair long amplicons. After purifying the LM PCR product with a column based gel extraction kit, measure the concentration using a UV spectrophotometer before proceeding to library preparation. Also, evaluate the length of the LM PCR products using a bioanalyzer instrument as per the manufacturer's instructions.
Prepare the library by setting up an indexing PCR. For each PCR, use 150 to 170 nanograms of purified LM PCR product, in different index combination for each sample. Perform the PCR in a thermal cycler with heated lid as indicated.
Following the PCR, dilute the purified library to 10 nanomolar with 10 millimolar tris HCL. Next, mix five microliters of diluted pool with five microliters of 0.2 newtons sodium hydroxide in a new 1.5 milliliter tube. Vortex briefly, spin down and then incubate for five minutes at room temperature to denature the libraries.
Place the tubes of denatured pool on ice and 990 microliters of pre-chilled HT1 hybridization buffer. Then aliquot 300 microliters of denatured pool into a new 1.5 milliliter tube. And add 300 microliters of pre-chilled HT1 to obtain a ten picomolar final library pool.
In parallel, mix 10 nanomolar of fixed control library in a two microliter volume with 3 microliters of 10 millimolar tris HCl in a 1.5 milliliter tube. Add five microliters of 0.2 newtons sodium hydroxide and vortex briefly. After spinning down, incubate for five minutes at room temperature to denature the diluted fix.
After the incubation, place the tube on ice and add 990 microliters of pre-chilled HT1. Now, aliquot 100 microliters of denatured fix in a new 1.5 milliliter tube and add 300 microliters of pre-chilled HT1 to obtain a 10 picomolar final concentration of fix. Finally, in a new 1.5 milliliter tube, mix 510 microliters of denatured library pool with 90 microliters of denatured fixed library, thus obtaining a final 10 picomolar pool with 15%of fix control to be sequenced.
This image shows the analysis of 3498, 2989, and 4103 clusters of recurrent MLV integration sites in hematopoietic stem progenitor cells, erythroid progenitors and myeloid progenitors respectively. In each cell population, greater than 95%of MLV clusters overlapped with epigenetically defined enhancers and promoters. Cell specific MLV targeted regions were highly acetylated and associated with cell specific expression of the targeted gene.
Examples are the promoter of HSP specific SPINK2 gene. The locus control region containing potent enhancers of the erythroid specific beta-like globin genes. Enhancers located upstream of the NPP specific LYZ gene.
Luciferase reporter assays validated the subset of cell specific MLV targeted enhancers in EPP and MPP. Erythroid specific MLV targeted regions had higher activity in EPP than in MPP and vice versa, confirming that MLV identifies cell specific enhancers possibly controlling the expression of nearby genes. Once mastered, this technique can be done in on week if it's performed properly.
While attempting this procedure, it's important to remember to maximize the frequency of transduced cells for optimal integration site retrieval. After watching this video, you should have a good understanding of how to map viral integration sites by first adjusting the genomic DNA then ligating the genomic fragments with a DNA linker followed by amplifying the host genome viral junction via LM PCR and lastly deep sequencing the amplicon library.
