This work was supported by the Georgia Cancer Coalition grant-R9028.

Rationale
Exploiting the use of Thermo Scientific Dharmacon oligos, 50 to 80-mers, here we present a procedure, based on a gene correction assay, by which oligos can transfer genetic information to genomic DNA of yeast cells, following annealing to the target chromosomal DNA. Successful targeting by oligos is scored by the appearance of yeast colonies displaying the expected phenotype. If the desired genetic modification is carried within one or more ribonucleotides incorporated in the oligo sequence, the genetic information flows directly from the RNA tract to the chromosomal target DNA, thus, an RNA/DNA hybrid forms at the chromosomal level during the targeting process. The ribonucleotide/s of a Thermo Scientific Dharmacon RNA-containing oligo can serve as template for gene targeting via DNA repair synthesis or can be embedded into the DNA and serve as template during DNA replication. In these experiments we use the yeast /Saccharomyces cerevisiae/ eukaryotic model system; however, a similar approach can also be applied in other organisms or cell types. In this video, we use a Thermo Scientific Dharmacon oligo designed with homology to a target yeast genomic locus and containing one ribonucleotide in the middle to correct a mutation in the target gene. After transformation with the RNA-containing oligo, we observe the correction of the targeted site at a certain frequency, which indicates that the RNA tract of the oligo could be incorporated into the chromosomal DNA and serve as template for DNA synthesis during DNA replication. The genetic information carried within the RNA tract is stably transmitted to the following cell generations. Here we describe the steps of yeast cell transformation by the Thermo Scientific Dharmacon RNA-containing oligo and the approach to detect the RNA information transferring into chromosomal DNA.
A. Design of the RNA-containing Oligo used in this Experiment
We have designed an RNA-containing oligo to correct a genetic defect in the yeast S. cerevisiae chromosomal DNA at the trp5 locus. The yeast strain used in the protocol contains a mutant trp5 gene with a two-base deletion and one nonsense mutation (Figure 1A). Such yeast strain is a tryptophan auxotrophic mutant and does not form colonies on media without tryptophan. The DNA sequence of the TRP5 gene is available at the Saccharomyces Genome Database (http://www.yeastgenome.org/). The Thermo Scientific Dharmacon RNA-containing oligo used in this protocol is a 65-mer with a 2-base DNA insertion, designed to correct the deletion mutation and one ribonucleotide designed to correct the nonsense mutation of the trp5 allele (Figure 1A). The sequence of the oligo is designed as follows: 
 5'-AAAAGGGTTTTGATGAAGCTGTCG-CG-GATCCCACATTCTG-rG-GAAGACTTCAAATCCTTGTATTCT. This oligo is synthesized by Thermo Scientific Dharmacon (Lafayette, CO) at 200 nM scale, and is desalted, deprotected and used without PAGE purification.
B. Preparation of the RNA-containing Oligo for Yeast Transformation (modified from Storici et al., 2007 1)
<ol>
 <li>Wipe materials that will be used for the experiment including oligo tubes, pipettes, vortex, racks, experimental area and gloves worn by the investigator with RNase decontamination solution to remove potential RNase contamination before everything starts. Use RNase-free water, chemical reagents, tubes and pipette tips in all steps. Every step in this experiment should be RNase-free.</li>
 <li>Resuspend the Thermo Scientific Dharmacon RNA-containing oligo received from the company to 250 pmoles/&mu;l stock solution with RNase-free water and vortex vigorously to dissolve the pellet. Store at -80&deg;C.</li>
 <li>Immediately before the transformation, thaw the RNA-containing oligo on ice and dilute to 50 pmoles/&mu;l with RNase-free water in RNase-free tubes. Each transformation requires 1 nmole of the RNA-containing oligos.</li>
 <li>Denature a chosen amount of the RNA-containing oligos on the 100 &deg;C heat block for 2 min to eliminate secondary structures of the oligos.</li>
 <li>Immediately after denaturation place the tube on ice. Keep on ice till transformation.</li>
 <li>As control in the experiment a corresponding DNA-only oligo is used. This oligo is thawed from -20 &deg;C and prepared for the transformation as the RNA-containing oligo, as described above.</li>
</ol>
C. Transformation of Yeast Cells using the RNA-containing Oligo
<ol>
 <li>Inoculate 5 ml of rich YPD liquid medium with the trp5 mutant yeast cells and grow at 30 &deg;C overnight (see Materials).</li>
 <li>Transfer 1.5 ml of the overnight culture into 50 ml of YPD liquid medium.</li>
 <li>Incubate cells in a 30 &deg;C shaker (225 rpm) for 4h.</li>
 <li>Prepare Solution 1 and Solution 2 immediately before transformation in RNase-free tubes (see Materials).</li>
 <li>Transfer the cell culture to a 50 ml RNase-free tube and spin at 3000 rpm, corresponding to 1562 g, for 2 min. The pellet of the cell precipitation is approx. 0.3 cm3.</li>
 <li>Remove the supernatant and wash cells with 50 ml of RNase-free water and spin at 3000 rpm for 2 min.</li>
 <li>Repeat step 6 for 5 times to get rid of the culture medium and RNases that could be present in the medium as much as possible.</li>
 <li>Remove the supernatant and resuspend cells in 5 ml of Solution 1 and spin at 3000 rpm for 2 min.</li>
 <li>Remove supernatant and resuspend cells in 250 &mu;l of Solution 1. This amount of cells is sufficient for 7-8 transformations. </li>
 <li>Aliquot 50 &mu;l of the cell suspension in RNase-free microcentrifuge tubes, add 20 &mu;l of RNA-containing oligo working solution (1 nmole), or 20 &mu;l of DNA-only oligo working solution (1 nmole), or 20 &mu;l of sterile water with no oligo for the negative control. Then add 300 &mu;l of Solution 2 for each transformation reaction. There is no need to add salmon sperm DNA in the process of transformation, as the oligos act as carrier themselves. </li>
 <li>Vortex vigorously to mix components homogenously.</li>
 <li>Incubate transformation reactions at 30 &deg;C for 30 min in a shaker.</li>
 <li>Heat shock at 42 &deg;C for 15 min. </li>
 <li>Spin down cells at 5000 rpm, corresponding to 2236 g, for 4 min.</li>
 <li>Remove supernatant and resuspend cells in 100 &mu;l of sterile water.</li>
 <li>Take an aliquot of this cell suspension and dilute it with sterile water by 100,000 fold and plate cells on one YPD plate using approx. 15 sterile glass beads and incubate at 30 &deg;C for 2 days.</li>
 <li>Plate all resuspended cells from each transformation reaction on one Petri dish of synthetic complete solid medium without tryptophan (SC-Trp) using approx. 15 sterile glass beads and incubate at 30 &deg;C for 4-5 days (Figure 2).</li>
</ol>
D. Analysis of Gene Correction by the RNA-containing Oligo
<ol>
 <li>Count the number of colonies grown on the selective medium (Figure 2) as well as on YPD medium to calculate the gene correction frequency for the RNA-containing oligo, the DNA-only oligo and for the no-oligo control. Compare the number obtained. Spontaneous reversion rate of the trp5 alleles with the two-base deletions and the nonsense mutation is less than 10-9 in the used yeast strain, thus we expect no colonies formation on the selective medium when no oligos are added to the cells.</li>
 <li>Streak out several randomly picked transformant colonies onto YPD medium to obtain single colony isolates. Wait two days for colony growth, then take several (at least 5) single colonies and make patches on YPD and on selective medium.</li>
 <li>Design a pair of primers to amplify the region (250-1,000 bp) targeted by the RNA-containing oligo by colony PCR (Figure 1B). The procedure for colony PCR is as follows (modified from Storici and Resnick, 2006 2).
 <ol>
 <li>Resuspend cells (approx. 1 mm3) taken from the individual patches in 50 &mu;l of water and add 1 unit of lyticase. Incubate at room temperature for 10 min, followed by incubation in a heat block at 100 &deg;C for 5 min to break the cells and release genomic DNA in solution.</li>
 <li>PCR conditions: The PCR reaction includes 10 &mu;l of the cell resuspension solution, 50 pmoles each of forward and reverse primers, 1 &mu;l of 10 mM dNTPs, 1 unit of Taq polymerase, 5 &mu;l of 10x buffer and is adjusted with sterile water to a final volume of 50 &mu;l. The PCR program is 3 min at 95 &deg;C; 30 cycles of 30 s at 95&deg;C, 30 s at 55 &deg;C, and 1 min at 72 &deg;C; a final extension time of 7 min at 72 &deg;C; then samples are kept at 4 &deg;C. An extension time of 1 min/kb is assumed for this reaction.</li>
 <li>Following PCR, samples are run on a 1% or 2% (depending on the expected size of the PCR product) agarose gel for observation of the PCR product (Figure 3). </li>
 </ol>
 </li>
 <li>If the genetic information transferred by the RNA-containing oligo generates a new restriction site in the yeast genomic target region (Figure 1B), it is possible to verify the correct transfer of information by digesting the PCR product with the appropriate restriction enzyme. If no restriction site is generated by the RNA-containing oligo, go to step 6. Digest PCR products using a specific restriction enzyme. The digestion reaction includes 6 &mu;l of PCR product, buffer, BSA (may not be needed for some enzymes, see instruction for the enzyme used), 0.5 &mu;l of restriction enzyme, and sterile water to 15 &mu;l. Samples are incubated for 1 h at the temperature specific for the enzyme used. </li>
 <li>Run an undigested sample together with the digested samples on the same row of a 2% agarose gel to observe the genetic modification transferred by the RNA tract of the RNA-containing oligo (Figure 3). </li>
 <li>Purify PCR products by using a PCR purification kit and prepare them for DNA sequencing. Submit samples for sequencing with the same primers used to amplify the product.</li>
 <li>Analyze DNA sequencing results using software that allows alignment of multiple sequences with a chosen reference sequence (Figure 4).</li>
</ol>
E. Alkali Treatment of the RNA-containing oligo (Figure 5)
<ol>
 <li>For each reaction, add 1 nmole (4 &mu;l of 250 pmoles/&mu;l stock solution) of the RNA-containing oligo, or DNA-oligo into a 1.5 ml tube.</li>
 <li>Add 4 &mu;l of 1 M NaOH for hydrolysis, or alternatively add 4 &mu;l H2O as negative control, and incubate at 65&deg; C in a water bath for 1 h. Then move from the water bath to ice.</li>
 <li>Neutralize with 2 &mu;l of 1.2 M HCl, 4 &mu;l of 1 M Tris-HCl, and 4 &mu;l of H2O, or alternatively 6 &mu;l of H2O and 4 &mu;l of 1 M Tris-HCl for negative control. Keep on ice till transformation.</li>
</ol>
<img src="/files/ftp_upload/2152/2152fig1.jpg" alt="Figure 1" /> Figure 1. Schematic diagram of the defective trp5 gene and of the TRP5 allele corrected by the RNA-containing oligo. A) The trp5 mutant gene contains a 2-base deletion (black triangles) and 1-base nonsense mutation (asterisk). The single-strand RNA-containing oligo with 2-base insertion (blue loop) and 1-RNA base substitution (red rectangle) generating a Van91I restriction enzyme site (shown by the bracket) is transformed into yeast cells to correct the genetic defects of the trp5 gene. B) After the TRP5 gene is repaired by the RNA-containing oligo (corrected bases are indicated as blue rectangles), the Van91I site is generated into the TRP5 gene. A 278 bp fragment including only one Van91I restriction site in TRP5 gene is PCR amplified by a pair of primers (P1 and P2). The 177 bp and 101 bp fragments generated after digestion of the P1 and P2 PCR product by Van91I are also shown.
<img src="/files/ftp_upload/2152/2152fig2.jpg" alt="Figure 2" /> Figure 2. Transformation result with the RNA-containing oligo. A) Yeast cells transformed with no oligo do not form any colony on the SC-Trp medium, see plates a-b. B) Plates c-g show yeast colonies growing on SC-Trp after cells are transformed with 1 nmole of the RNA-containing oligo. C) Plates h-l show yeast colonies growing on SC-Trp after cells are transformed with 1 nmole of the corresponding DNA-only control oligo.
<img src="/files/ftp_upload/2152/2152fig3.jpg" alt="Figure 3" /> Figure 3. Detection of genetic information transfer from the RNA-containing oligo to yeast chromosomal DNA by restriction digestion of the PCR product amplifying the targeted genomic region. 2% agarose gel electrophoresis of PCR samples amplified by P1 and P2 and digested with Van91I restriction enzyme. Lanes 1, 8 and 15, DNA ladder with sizes of 100, 200, 300, 400 and 500 bp indicated on the left; lane 2, PCR product of trp5 locus amplified from the genomic DNA of the trp5 mutant strain; lane 3, PCR product amplified from genomic DNA derived from a Trp+ colony targeted by the DNA-only oligo; lane 4-7, PCR products amplified from genomic DNA derived from Trp+ colonies targeted by the RNA-containing oligo; lane 9 to 14, Van91I restriction digestion of the PCR products from lanes 2 to 7. The presence of the uncut PCR product bands in lanes 10 to 14 can be explained by partial digestion by Van91I at the TRP5 locus (CCACATTCTGG). Considering that the cutting site for Van91I (CCANNNN NTGG) can have multiple sequences, the site generated in TRP5 may not be the most optimal target for the enzyme. In fact, following DNA sequencing of all the above PCR products we detect no additional changes beyond those carried by the oligos (see Figure 4). The 278 bp PCR band amplified by P1 and P2 primers and the digestion product bands by Van91I of 177 bp and 101 bp are shown by the arrows on the right.
<img src="/files/ftp_upload/2152/2152fig4tmb.jpg" border="0" alt="Figure 4Thumb" /> Figure 4. DNA sequencing results showing gene correction by the RNA-containing oligo. A) DNA electropherogram of the genomic region targeted by the RNA-containing oligo. The G in the DNA sequence (blue boxed) derives from the rG on the RNA-containing oligo. Also boxed is the insertion of the CG bases. Sequencing results from all other PCR products are also as clear as this one, with fluorescent signals well above the background. B) Sequences of the TRP5 region of the Trp+ transformants targeted by the DNA-only oligo (T1) and the RNA-containing oligo (T2-T5) match in the consensus sequence on the top and are compared with that of the trp5 mutant cells before targeting by the oligos (Genomic DNA, red shaded). The repair RNA-containing oligo on the bottom has two-base DNA insertion and one-base (G) RNA substitution marked in red. The regions boxed in blue with yellow shade show that the RNA-containing oligo as well as the DNA-only oligo precisely corrected the deletion mutation and nonsense mutation in all tested samples. The dashed lines mark the position of the RNA-containing oligo sequence. Please <a href="https://www.jove.com/files/ftp_upload/2152/2152fig4.jpg">click here</a> to see a larger version of figure 4.
<img src="/files/ftp_upload/2152/2152fig5.jpg" alt="Figure 5" /> 
 Figure 5. Alkali treatment prevents gene correction by the RNA-containing oligo. The transformation frequency by the RNA-containing oligo (R) is shown in the red bar and that by the DNA-only oligo (D) is shown in the blue bars. The error bars represent the standard error of the mean for 3 independent transformations for each oligo. The DNA-only oligo displays similar transformation frequency without and with NaOH treatment. Differently, transformation frequency by the RNA-containing oligo drops to 0 following treatment with NaOH. Therefore, the preparation with the RNA-containing oligo is not contaminated with DNA-only oligo, thus, the observed transformation frequency is specific to the RNA-containing oligo.

<ol>
	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

The video production for this article was sponsored by Thermo Fisher Scientific, which produces reagents and instrument used in this publication.

The fact that RNA can transfer genetic information directly to genomic DNA in cells was discovered exploiting the use of synthetic RNA-containing oligos (Dharmacon synthesized oligos) 1. It was the proof of principle that cells can use RNA-containing molecules or RNA-only sequences as templates for DNA synthesis. The use of RNA-containing oligos not only led to the demonstration that genetic information can be transferred directly from RNA to genomic DNA without the need of a reverse transcribed DNA copy intermediate, but also to the proof that RNA can be used as homologous template in the repair of a DNA damage 1,3. The oligos can be made of RNA-only or contain just a single ribonucleotide embedded in a DNA sequence, as in the example we presented here. The RNA-containing oligo has one embedded ribonucleotide designed to correct the nonsense mutation in the genomic defective trp5 allele. The transfer of genetic information from the ribonucleotide to the genomic DNA is revealed by a change in phenotype (cell growing on the medium lacking tryptophan) of the targeted cells. The frequency of gene correction obtained with the RNA-containing oligo is measured and compared with that of a control DNA-only oligo. By performing this gene correction assay in different cell backgrounds, where we mutate various yeast genes, we can detect which factor/s specifically affect targeting by the RNA-containing oligos. Thus, we can reveal mechanisms how cells regulate the stability of RNA/DNA hybrids. By simply designing different variants of the RNA-containing oligos we can determine the likelihood for a particular RNA tract to serve as template in DNA modification and we can determine the stability of specific RNA sequences embedded into DNA. Moreover, we can identify what are the preferred in vivo substrates for factors interacting with RNA/DNA hybrids.
While several in vitro studies, mainly utilizing short RNA-containing oligos, have been conducted to characterize the function of factors that can recognize RNA in a hybrid with DNA, such as the RNase H enzymes 4, the in vivo functions of RNases H as well as the identity of other proteins that could affect RNA/DNA hybrid stability remain mostly unknown. The possibility to utilize RNA-containing oligos of a significant length (50 to 80-mers) and optimal quality (such as Thermo Scientific Dharmacon RNA-containing oligos) has open the way to examining a wide range of molecular processes directly in vivo in the cells of interest. As shown in this video, transformation using RNA-containing oligos requires essentially only a few additional steps compared to transformation using DNA molecules, to prevent degradation by RNases. Thus, transformation using RNA-containing oligos is not limited to the yeast system, but can be applied to any organism or cell type where transformation by DNA oligos is proficient.
In conclusion, targeting cells by RNA-containing oligos provides the opportunity to generate RNA/DNA hybrids and ribonucleotides embedded in DNA in vivo in cells. The stability, function and consequences of these in vivo generated RNA/DNA hybrids can be analyzed and characterized, potentially uncovering unknown mechanisms of DNA repair and revealing novel strategies for gene targeting.

A. Transformation reagents and media (modified from Storici and Resnick, 2006 
<ol>
 <li>YPD (Yeast Peptone Dextrose): For 1 L, 10 g yeast extract, 20 g soy peptone, 20 g dextrose. Add 15 g agar to make YPD solid media. Autoclave before use. Store at room temperature.</li>
 <li>Solution 1: 0.1 M of lithium acetate. Prepare immediately before transformation. Solution 1 is a working solution, thus it is prepared directly from the powder. No stock solution is made. Keep at room temperature. LiAc increases the yeast cell wall permeability to DNA. </li>
 <li>Solution 2: 0.1 M of lithium acetate and 50 % of polyethylene glycol 4000. Also solution 2 is a working solution and it is made directly from the powder. No stock solution is prepared. Keep at room temperature. PEG deposits oligos onto yeast cells.</li>
 <li>RNA-containing oligos (Thermo Scientific Dharmacon), 50-80-mers, desalted, deprotected and non-purified. Resuspend to 250 pmoles/&mu;l. Store at -80&deg;C.</li>
 <li>DNA-only oligos, 50-80-mers, desalted and non-purified. Resuspend to 50 pmoles/&mu;l. Store at -20&deg;C.</li>
 <li>SC-Trp (Synthetic complete media lacking tryptophan) solid media.</li>
 <li>0.5 cm diameter glass beads, sterilized by autoclaving. </li>
 <li>RNase-off: RNase decontamination solution.</li>
 <li>DNase/RNase-free, sterile centrifuge tubes.</li>
 <li>DNase/RNase-free, sterile conical tubes.</li>
 <li>DNase/RNase-free, sterile aerosol pipette tips with ZAP: 1-200 ml, 100-1000 ml.</li>
</ol>
B. Colony PCR materials.
<ol>
 <li>DNA primers, desalted and non-purified. Dissolve in sterile water to 50 pmoles/&mu;l. Store at -20&deg;C.</li>
 <li>Taq DNA polymerase, buffer, dNTPs.</li>
 <li>PCR tubes.</li>
</ol>
C. PCR purification.
<ol>
 <li>PCR purification kit.</li>
</ol>
D. Gel Electrophoresis.
<ol>
 <li>Agarose.</li>
 <li>1 x TBE running buffer (45 mM Tris-borate and 1 mM ethylenediamine tetraacetate) diluted from 10x TBE. </li>
 <li>Prestained molecular weight marker.</li>
 <li>DNA loading dye.</li>
</ol>
E. Restriction digestion.
<ol>
 <li>Restriction enzymes, 10x buffers, BSA.</li>
</ol>
F. Alkali treatment for the RNA-containing oligo.
<ol>
 <li>1 M of NaOH solution.</li>
 <li>1.2 M of HCl solution.</li>
 <li>1 M of Tris-HCl, pH 7.4 solution.</li>
</ol>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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26.8K Aufrufe.  Georgia Institute of Technology. Diese Arbeit zeigt, wie ein RNA / DNA-Hybrid bei der chromosomalen Ebene bilden und zeigen Übertragung der genetischen Information von RNA, um genomische DNA in Hefezellen.

Serie: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Isolation of Genomic DNA from Mouse Tails

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

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The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

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The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

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Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...