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The overall goal of this procedure is to rapidly generate large homologous recombination vectors that can be used to target and manipulate endogenous oph loci. This is accomplished by first selecting a back clone, covering the genomic region of interest. The second step of the procedure is to clone homology arms into a homologous recombination vector.

The third step is to clone genomic DNA into the homologous recombination vector using gap repair. The final step is to replace the genomic region with the targeting cassette. Ultimately, these procedures will engineer a targeting vector for Enzo homologous recombination to generate unequivocal null mutations or tagging endogenous proteins in drosophila through the usage of recombining based techniques.

Additionally, the written manuscript also includes a protocol for how to mobilize these cassettes in vivo to generate a knockout or a tagged gene via knockin. The main advantage of this technique over existing methods is that it can easily be adopted for multiple targets in parallel, and it provides a means for manipulating the oph genome in a timely and efficient manner. To achieve this goal, our methods combine recombining and gateway technologies to generate the homo recombination vectors.

In contrast to conventional restriction inside Ligase cloning, these technologies are not limited by the size or sequence of the DNA, thereby facilitating the cloning process. While this protocol describes a method to generate a targeting cassette for gene knockout, it can also be used to manipulate any endogenous locus in virtually any desirable manner. This includes tagging a gene of interest, replacing a gene of interest with GAL four, or to generate protein truncation or reporter lines To acquire a back with the gene of interest, which is CG 3 2 0 9 5.

In this example, open a search for the gene@flybased.org. To get the list of backs. Look under the stocks and reagent section and check the section entitled genomic clones.

From the available options, find a clone that includes at least 10 kilobases upstream and five kilobases downstream of the gene of interest. These regions are used as homology arms. This can be done by aligning the five prime end and the three prime end sequences of the back against the drosophila genome.

Alternatively, clones in the Pac-Man vector can also be found@pacmanfly.org. Now select primers to amplify the 500 base pairs at the end of each of the homology arms. These fragments called LA and RA are used to recombine the genomic region between the homology arms into the Pacman.

KO 1.0 plasmid. Make certain that the 500 base bear homology arms do not contain BAM H one restriction sites, as these will be digested to linearize the plasmid. Set up two individual PCR reactions to amplify the LA and the RA fragments for the LA fragment into the forward primer, incorporate a TTV one and ISC one sequences in the reverse primer.

Incorporate AB MH one site seven bases three prime to the complementary sequence corresponding to the opposite homology arm. For the RA fragment include AB MH one site seven bases three prime to the complementary sequence corresponding to the opposite arm in the forward primer and the at TB two, and I sce E one sequences in the reverse primer as a template. Use one microliter of back DNA from a dirty mini prep, or half a microliter of back containing bacteria that was cultured overnight and boiled in this and all subsequent PCRs.

Make sure to use a DNA polymerase with proofreading capability. This is not however necessary for the PCR checks. Run the PCR samples on a 1%aros gel and extract the products using a XMO clean DNA recovery kit.

Elute the DNA with 10 microliters of sterile water. Next, using 10 to 30 nanograms from each amplified DNA. Perform a splicing by overlapping extension PCR.

This usually uses about one 20th of the purified PCR products. Now using a BP reaction following the manufacturer's protocol, clone the LARA fused fragment into the Pacman KA plasmid. This plasmid can be requested from the Boost Truck Lab inoculate SW 1 0 2 cells containing the back of interest into four milliliters of LB plus the appropriate selection antibiotic in this case, 25 micrograms per milliliter.

Chloramphenicol grow the culture at 32 degrees Celsius overnight at 250 RPMs. Be cautious that the temperature does not exceed 32 degrees Celsius or the recombination machinery of the cells will be activated the following day. Digest 0.4 micrograms of the Pac-Man KO containing the LA and RA with approximately 20 units of bmh.

One in a 25 microliter reaction at 37 degrees Celsius. Run a long reaction of at least three hours as this is critical for eliminating false positives. Next, inoculate a milliliter of the saturated SW 1 0 2 back culture in 44 milliliters of LB with chloramphenicol.

Grow it at 32 degrees Celsius with shaking to an OD 600 of 0.2 to 0.3, which usually takes between 60 and 90 minutes. Also start a parallel culture as a non heat shock to negative control. The appearance of many colonies on this control usually indicates transformation with undigested plasmid.

While the culture is growing. Run the BAM H one restricted plasmid on a gel, then gel extract and dilute the DNA in 10 microliters of warmed water, but definitely not buffer. At up to 55 degrees Celsius, salt and buffers will foul.

The electroporation heat shock the culture at 42 degrees Celsius for exactly 15 minutes in a water bath, but leave the non heat shock control culture at 32 degrees. Then immediately transfer both cultures to chilled 50 milliliter conical tubes and let them sit in an ice water slurry for five minutes. Keeping the cells at low temperatures is crucial to their competence.

Never resuspend the cells by repeated pipetting or vortexing. Now centrifuge the cultures discard the supernatant and wash the pellet in ice cold 10%glycerol first, add five milliliters of glycerol. Next resus.

Suspend the cells with tapping and swirling, and then add 20 more milliliters of glycerol. Repeat the centrifugation and resus suspension in 10%glycerol twice more, and then centrifuge the cells once again. At this point, the cell pellet may be very loose and care should be taken when discarding the supernatant.

Now suspend the pellet in one mil of cold, 10%glycerol and transfer the cells to a 1.5 milliliter tube. Centrifuge the cells for 30 seconds at 12, 000 Gs at four degrees Celsius and discard most of the supernatant leaving behind about 90 microliters of cells in glycerol. At this point, the cells can be stored at negative 80 Celsius.

However, this may decrease their competence. Now add two microliters or 50 nanograms of the BAM H one digested plasma to the cells and stir them gently Using a pipette tip. Transfer the mixture to a one millimeter electroporation, qve, and ate the cells.

Next, add 300 microliters of SOC and let the cells recover for two hours at 32 degrees Celsius without shaking. After the recovery period, plate the cells on LB agar with 50 micrograms of ampicillin per milliliter solution. Then incubate the cells at 32 degrees Celsius for 24 to 30 hours the next day.

Screen for proper gap repair colonies by PCR. Use T three and RA check primers for the RA fragment and T seven and LA check primers. For the LA fragment, it is critical to test both arms since incorporation of one arm but not the other is sometimes observed.

The check primers must be designed 100 to 200 base pairs towards the targeted region. If subsequent steps fail, these primers can be validated using neighboring primers and the original back as a template. The positive colonies from the PCR will show a band that runs a 800 to 1000 base pairs.

Grow up the positive colonies at 32 degrees Celsius in four milliliters of media With ampicillin overnight, a frozen stock of these cells can be made for later use. Begin by amplifying the RFP can knockout cassette from linearized plasmid using the appropriate primers. Then gel purify the product and ute it in 20 microliters of sterile water.

The RFP can cassette plus the homology arms should run around three kilobases inoculate. One milliliter of the saturated SW 1 0 2 Pac-Man KO culture into 44 milliliters of LB with ampicillin. Grow it up at 32 degrees Celsius on a shaker to an OD 600 of 0.2 to 0.3.

Also include an identical sample for the non heat shock negative control. After the cultures reach the desired density, purify them for electroporation as described in the previous section. Next electro about 100 nanograms of the targeting cassette into 90 microliters of the electro competent cells using the same method previously reported.

After the cells have recovered for two hours, plate them on LB agra with ampicillin and can mycin incubate the plates for 24 to 30 hours at 32 degree Celsius. The next day, grow five milliliter cultures from five different colonies at 32 degrees Celsius overnight. On the following morning, perform a dirty mini prep and run half of the obtained DNA on a 1%agros gel to look for the presence of a low molecular weight plasmid.

There should not be any plasmid that runs below 12 kilo basises as seen in lane two of this gel. Now make a one to 500 dilution of the DNA from a positive clone and use one microliter of the diluted DNA to ate into 50 microliters of Tmax epi 300 cells plate the recovered epi 300 cells in LB agar with ampicillin and can mycin and incubate them for 18 to 24 hours at 37 degrees Celsius on the following day. Inoculate a single colony in 10 milliliters of lb plus antibiotics and grow it up overnight on the next morning from the saturated culture seed 100 milliliters of lb.

Add the appropriate antibiotics and add 100 microliters of 1000 x copy control solution. Incubate the culture at 37 degrees Celsius for five to six hours with shaking later that day. Perform a maxi prep and verify the insertion of the cassette in the correct site by sequencing.

Now design and perform a restriction enzyme digest test using a clone with correct sequencing results to characterize the combier vector. In this example, the CG 3 2 0 9 5 targeting vector was serially digested with PAC one, ASC one, BH one, and a T two. The appearance of all the predicted bands and absence of any incorrect bans confirms that the targeting vector has been correctly combier.

The vector may now be transformed into flies. Expressing recombinase transformed flies can be selected from their progeny. Note that large vectors are sensitive to shearing.

We avoid freeze thaw cycles and prepare DNA fresh for injection. The resulting transgenic flies contain the targeting construct in a predefined landing site, but not yet in the endogenous locus in the genome. The final step of replacing the endogenous locust does not require any further molecular biology, but only published standard drosophila methods that include heat shock mediated mobilization of the targeting cassette and a fly screen for reintegration of the targeting cassette away from the original landing site.

These methods are described in recent publications. Amplification of the LA and RA homology arms should produce 500 base pair products and the P-C-R-S-O-E reaction should yield a one kilobase product. The BP reaction is typically very efficient.

Bacterial transformation of its product usually yields five to 100 colonies. Nearly all the colonies tested with a PCR check show the expected product during the first round of re commandeering expect to get 20 to 40 colonies after transforming the digested Pacman KO 1.0 containing the LA and RA arms into SW 1 0 2 cells containing the back of interest. 40 to 60%of these clones will carry the desired recombination product.

In this example, about 60 colonies were obtained and the PCR check showed about 60%of the colonies carried the desired recombination product. The second round of recombining is typically much more efficient. Normally three to 50 colonies are observed after transformation with 90 to 95%containing the desired construct Plasmid below 12 kilobases identifies false positive colonies such as clone two.

In this example. While attempting this protocol, it's important to remember that in bacteria recombination of large fragments of DNAs are very inefficient. Therefore, to obtain optimal results is crucial to prepare high quality competent cells and to use fresh DNA.

After watching this video, you should have a good idea of how to generate a targeting vector for N out homologous recombination in Drosophila. This vector could be used to produce any clean, molecularly defined manipulation of single genes at their endogenous loci.