Testing The Selection Cassette For Gene Targeting Biology Essay

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Gene targeting approaches in somatic cells are used to generate 'knockouts' and 'knock-ins' that makes it possible to study the function of targeted genes or introduced mutations. To increase the efficiency of gene targeting, targeted cells were separated from non-targeted ones by using selection based on neomycin resistance and the expression of HA tag on the surface of targeted cell. We investigated colorectal cancer cell HCT-116, in which PRPS 1 gene has been knocked out by homologous recombination. In this present study, the cells that had successfully undergone gene targeting were resistant to G418 antibiotic, because of the presence of neomycin (Neo) resistant gene as selectable marker. To confirm neomycin resistance, antibiotic sensitivity test was carried out and survived clones shows the presence of selectable marker. To further validate the construct, we used cells previously treated with Cre recombinase to remove the Neo gene, as to test the cells were able to survive in the presence of antibiotics. Furthermore as the construct contains HA tagged Neo, we used anti HA-antibodies to detect fusion protein on western blot. No specific signal was detected in Western blotting; however a band was seen after immunoprecipitation although the molecular weight differed from the expected one. Further studies are required in modifying the sequence of the construct so as to detect HA tag proteins.

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Introduction

The important applications of gene targeting approaches in somatic cells are generation of 'knockouts', that determines the loss-of-function of that particular gene, and 'knock-ins', that includes alternation of single or several bases1. Gene targeting is a genetic technique that uses homologous recombination to modify an endogenous gene. These modifications can be point mutations, removal of exons and addition or deletion of a gene. Homologous recombination is a genetic recombination between DNA sequences in the chromosome and cloned DNA sequences that allows the transfer of any modification in the cloned gene into the genome of a living cell2. The introduction of exogenous DNA in mammalian cells often results in random integration at higher levels than gene targeting, thus hindering gene therapy applications3. Gene targeting can be used to improve the efficiency of gene correction in gene therapy by the use of homologous recombination4. Also gene targeting by homologous recombination helps in analysing multidrug resistance to chemotherapy in cancer patients by enhancing the gene expression5.

Although gene targeting by homologous recombination is a powerful tool to control the genome and to study the gene functions, there are other methods that can be applied to inactivate genes in cells. Among them is RNA interference (RNAi) is routinely used to knock down gene expression. Homologous recombination can be used as gene targeting in human cells lines including the HCT116 colon cancer cells6. The other methods for constructing gene targets through knockouts are by the used of inverse-PCR (IPCR)7.

To be able to scale up the process of enriching targeted cells from non- targeted ones, we used the strategy for separation based on growing cells in presence of antibiotics and sorting out cells expressing tagged HA protein on its surface. Previously generated PRPS-1 (phosphoribosyl pyrophosphate synthetase 1) knocked out HCT-116 cell was used to test the cassette. Our construct contains selectable marker which is the antibiotic resistant gene Neo fused to Immunoglobulin kappa (Ig κ) leader sequence, Internal ribosomal entry sequence (IRES) sequence, Hemagglutinin A (HA) tag and trans-membrane domain from insulin receptor and a LoxP site as flanking region.

Figure 1: Elements of the selection cassette, IRES is Internal Ribosomal Entry Sequence, Ig κ is Immunoglobulin kappa, HA is Hemagglutinin A tag, Ins R is an Insulin Receptor and Neo is a Neomycin gene. loxP is used as flanking regions8 (modified from Weide T et.al BMC Microbiology).

The elements for our construct are shown in Fig 1. The functions of the individual elements are; LoxP site, it is used to create a specific recombination which involves the targeting of a particular sequence of DNA and splicing it by an enzyme Cre recombinase. The presence of two loxP sites directly results in the excision of the DNA between the sites9. IRES is used to increase the translation efficiently by recruiting ribosomes, thus allowing protein synthesis from the middle of messenger RNA (mRNA)10. Ig κ acts as leader sequence is coupled with our fusion gene. The translation starts from this region onwards to give rise to fusion protein and it drives the fusion protein to out of the cell. HA tag, it's a protein tag which is expressed on surface of a targeted cells. By using specific antibodies to HA allows in detection of the fusion protein. Insulin receptor acts as trans-membrane domain; it anchors the fusion protein to cell membrane thus enabling the HA protein to be expressed outside the cell. Neomycin gene is an antibiotic resistant marker which the cells resistance to G418 antibiotic medium. This protein will be expressed inside the cell, thus making only the targeted cells resistance to the antibiotics.

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The targeted cells will have the HA tag which will be expressed outside the plasma membrane and the Neo gene will be present inside the cell. Thus the selection cassette is regarded as double selection of targeted cell over conventional gene targeting. By using specific antibodies targeting against HA, we can pull down the expressed cells and by growing in presence of antibiotics will facilitates only the neo gene carrying cells to be survived.

The presence of a selection cassette may interact with the endogenous gene expression at the targeted locus or its surrounding genes. To nullify these unwanted expressions, the marker gene cassette is flanked with loxP sequences, thus providing an option for the latter removal of the cassette with Cre recombinase6, leaving the targeted site as intact as possible. In this way it is possible to target the second allele, as the selectable marker is removed. Also recombinase-mediated cassette exchange allows sequential targeting at any locus and improves flexibility in making user-defined mutations11.

Materials and methods

Cell culturing

Parental HCT116 cells were grown in Complete McCoy's 5A media (supplied by Invitrogen) supplemented with 10% foetal bovine serum and 1% penicillin and streptomycin12, incubated at 370 C in 5% CO2. Previously generated PRPS1 knockout cells were grown in complete medium containing 400µg/ml Geneticin (G418). For sub-culturing, the medium from cultured flask was removed and cells were washed with Hank's Buffered Salt Solution (HBSS) and trypsination was carried out by treating with 0.25% trypsin solution and re- suspended cells were transferred into another flask with complete media.

Sensitivity to antibiotic media

To observe the viability of both, prior to Cre recombinase treated cells (pre Cre) and after Cre recombinase treated (post Cre) cells were cultured in McCoys media in the presence and absence of G418 in separate 6- well plates .Both post and pre Cre clones were grown in presence and absence of G418 in triplicates, incubated for 5 days at 370 C in 5% CO2. The medium was discarded; cells were stained with methylene blue (dissolved in 5% methanol) Solution was poured out, gently rinsed with double distilled water and plates were observed in Gel Doc™ XR+ imaging system (Bio Rad).

Transfection

Transfection was carried out in 6-well plates using pDisplay vector (supplied by Invitogen). A day prior to transfection roughly 700 000 cells (using spectrometry) were plated into each well. Next day, 5 μL lipofectamine was mixed with 0.5ml of media without FBS and antibiotics and 1 μg of pDisplay vector in 0.5ml of media without FBS and antibiotics were taken in 2 separate tubes left for 5 minutes at room temperature (RT). The 2 solutions were combined and allowed 20-minute incubation at RT. Cells was rinsed with medium without FBS and antibiotics before adding transfection solution. After 3 hours of incubation at 37°C, 2ml of complete McCoy was added. Cells were then grown for 24 hours at 37°C before analysis13.

Cell harvesting and Lysis

In order to measure the intracellular protein expression, the harvested cells were lysed using lysis buffer (100mM Tris-HCl, 150mM NaCl, 1% NP 40 and 1x Protease Inhibitor tablet {Roche}). Briefly, PRPS1 deleted HCT116 cells were harvested by trypsination using 0.25% trypsin, cell pellet was collected by spinning down the cell medium at 1500 rpm for 5 minutes and pellets were washed with 1x phosphate buffered saline (PBS) frozen at -200 C and 80µl of lysis buffer was added.

A day after after transfection with pDisplay cells were harvested. Briefly, cell lysing was done by removing media, washed with 1x PBS, and plates were frozen at -20o C. The cell lyses was done directly on to wells by adding 80µl of lysis buffer. Cell lysates were incubated on ice for 30 minutes with vortexing every 10 minutes. After incubation the tubes were centrifuged at 14000 rpm for 15 minutes and supernatant was collected.

Immunoprecipitation

Immunoprecipitation (IP) was carried out to enrich fusion protein from total lysate solution. From the cell lysate, 325µg of the protein concentration from 2 samples was taken to carry out immunoprecipitation, lysis buffer and 10µg (of 5µl) rabbit anti-HA antibody conjugated on agarose beads (Santa Cruz) was added and incubated for 4 hours at 4o C. After incubation the mixture was spun down at 3000 rpm for 1 minute. The pellet was washed with PBS and lysis buffer for 6 times alternatively with PBS and followed by lysis buffer, for each time repeating the centrifugation step. Care was taken while aspirating and discarding the supernatant. After the final wash the pellet was suspended in electrophoresis loading buffer (Invitrogen) and stored at -200C for overnight. The remaining 30µg of the proteins was used as input (ip) and pellet as IP was carried out for western blotting.

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Western Blotting

After cell lysis, protein concentration was measured using Bradford protein assay. 70µg of each sample was mixed with 8µl of loading buffer and samples were denatured at 700C for 10 minutes and subjected to NuPAGE 4~12% bis-Tris Gel electrophoresis (supplied by Invitrogen). The running conditions were at 180V for 45 minutes. Proteins were transferred onto nitro cellulose membrane using transfer buffer (25mM Tris base, 193mM Glycine, 20% Methanol). The electrophoresis condition was at 30V for 2 hours. After the transfer of proteins, the membrane was rinsed with 1x PBS and incubated with the appropriate primary and secondary antibodies. The primary antibodies were incubated overnight on shaker at 4oC. After incubation the membrane was washed with 1x PBS + 0.1% Tween solution for 1 hour, changing the solution for every 10 minutes. After washing, membrane was incubated with secondary antibody for 1 hour at RT on shaker, following incubation the membrane was washed with 1x PBS + 0.1% Tween solution for 30 minutes with changing the solution every 10 minutes.

The primary antibodies used were: mouse anti-HA (1:1000)(Covance), mouse α-tubulin (1:1000) (Abcam), goat β-actin (1:2500) {Santa Cruz}. The secondary antibodies were: corresponding HRP-conjugated (horseradish peroxidase) anti-mouse (1:5000) and anti- goat (1:5000). All the antibodies were dissolved in 3% bovine serum albumin (BSA).

Luminescence was detected by CCD Camera (Fuji Film) using developing solution (Lumi Light Western Blotting Substrate Solution), α-tubulin and β-actin was used as loading control.

Results

Sensitivity to antibiotics

Antibiotic sensitivity test was carried out to determine the presence of construct in the clones of PRPS-1 gene knocked out cells. As this results shows that, if a cell was able to survive in G418 medium indicates the presence of selection cassette which has neo resistant gene enabling cells to grow in medium containing antibiotics. By this we can enrich the targeted cells from non-targeted.

Previously generated PRPS1 knocked out cells was grown in G418 medium and the survived cells shows the resistant to neomycin, indicates the presence of neo gene in the construct. The clones 2, 4, 5, 6, 7, 8, 13, 20 survived in medium containing antibiotics. To further validate the construct, we used Cre recombinase mediated recombination that catalyses DNA excises between loxP sites, which in turn removes the selection cassette, making the cells sensitive to antibodies. Clones that have been treated with Cre recombinase were selected for analysis. To confirm, both post Cre and pre Cre clone 2 was grown in separate plates with presence and absence of antibiotic medium. The viability of cells was visualized by the help of methylene blue indicator. As methylene blue makes nuclei of live cells visible and thus it can easily distinguish between dead and live cells. (Fig 2)

Figure 2: Selection of clones. A) Pictorial representation14 showing strategy for Cre mediated recombination for removal of Lox P site in selection cassette by Cre recombinase. Here 1, 2, 3 and 4 are exons, the construct containing exon2 is introduced between exon 1 and 3 via homologous recombination. This image was taken from Access Engineering (McGraw-Hill). B) Cells were grown in -G418 medium, both post and pre Cre cells showed growth. Absence of G418 is insensitive to both post and pre Cre cells. C) Cells were cultured in +G418 medium, only pre Cre cells were able to survive in antibiotics indicating the presence of selectable marker neo gene whereas post Cre cells where dead showing the successful removal of cassette by Cre enzyme.

Western blotting

Western blotting was carried out to analyse the presence of fusion protein band on the membrane, which was expected to be 100 kDa. This approximate molecular weight was determined by calculating the number of bases in sequence multiplying by 0.11 (bp*0.11). The results of western blot did not show the expected band of our construct, or it may be a weak signal, that couldn't be detected because of background disturbance. The transfected vector pDisplay which was used as a positive control for antibodies was detected, which infers that both primary and secondary antibodies are accurate enough to detect HA proteins. Western blot analysis did not showed any difference between post and pre Cre clones except heavy and light chains of antibodies. But in the case of immunoprecipitated clone-5 (IP 2), there was weak signal corresponds to 70kDa. There was clear distinguished bands for heavy and light chains of the antibodies used which were corresponding at 50 kDa and 20 kDa respectively. (Fig 3).

Figure 3 A) Western blotting of all clones including transfected cell and control. No specific band of expected molecular weight was observed and pDisplay vector was detected inferring antibodies were able to detect HA protein. The control is a un-transfected cell. α-tubulin is used as loading control to check proteins were equally loaded. B) Western blot was done to differentiate between post and pre Cre clones and enriched HA proteins using IP. In clone-5 (IP 2), there was weak signal corresponds to 70kDa. IPs showed clear distinguishes of both heavy and light chains of the antibodies. 90% of total cell lysate was used for IP and remaining 10% as input (ip). β-actin was used as loading control.

Discussion

In this study the targeted cells from non-targeted were enriched by double selection process. As our construct contains neomycin gene, we cultured the cells in medium containing G418 as the survival cells shows the presence of our construct containing the neo gene. Antibiotics sensitivity test was carried out to validate the presence of cassette in the cells. The clones were separated based on their survival in the medium containing antibodies. Further this result was proved by using Cre mediated recombination. As the cassette contains the flanking region LoxP site, previously Cre treated and non Cre treated cells were separately grown in presence and absence of antibiotics. As the Cre recombinase mediates the removal of DNA in between the flanking region, thereby removing the construct within the LoxP sites. In absence of antibiotic medium, both prior to cre mediated (pre Cre) and after cre mediated (post Cre) showed the survival. As the presence or absence of the construct, did not produce any significant changes in their survival in the medium which was devoid of antibiotics. In the case antibiotic medium only the prior to cre mediated cells were survived showing the presence of neo gene in the construct, whereas the cre mediated cells were dead. Thus the post Cre cells shows the successful removal of the construct by the Cre enzyme and thus the neo gene. This result further strengthens the presence of the construct in pre Cre clones to survive in presence of antibiotics.

As the construct contains HA tag which is expressed on the surface of the targeted cells, anti HA antibodies were directed against the tagged proteins. Based on the sequence of the construct, approximate molecular weight of our fusion protein was determined. The lysate after separating on SDS gel, HA antibodies were directed to determine fusion proteins. We couldn't see signals in any of the survived clones. IP was carried out to enrich the fusion protein by using agarose conjugated HA antibodies. Here we were able to see weak signal on nitro cellulose membrane, but this signal was not corresponding to our expected weight.

Conclusion

In this study we had a positive result, that the clones of PRPS-1 gene knocked out cells which has our construct where growing in presence of medium containing antibiotic G418, indicating the presence of selectable marker neo gene. Prior to Cre recombinase mediated cells (pre Cre) survived in G418 medium whereas Cre mediated treated clones (post Cre) where dead in presence of antibiotics. From this, we can understand that our construct which contains the neo resistant gene have got incorporated in the cell which makes resistant to G418. As our cassette is a promoter-less sequence, the construct should have been incorporated in desired region driving the expression of neo gene to get transcribed by IRES, this transcribed neo gene is translated making the cells resistant to antibiotics. By removing the construct containing neomycin gene using Cre mediated recombinase makes cells sensitive to antibiotics and eventually died in presence of antibiotics.

However in western blotting, we didn't get the desired results, as our fusion protein was not detected on the membrane. As there was not band expected around 100 kDa, we assumed to be the molecular weight of our fusion protein based on the number of base pairs. There was a faint band in the case of immunoprecipitated clone-5 (IP 2) which corresponds to 70kDa. Maybe this band could be our desired protein as we have approximately calculated the molecular weight of our fusion protein based on the number of base pairs.

The reason for not detecting fusion protein could be of the endogenous promoter of PRPS-1 gene may not be strong enough to drive the expression of HA tag protein. Due to this weak expression, the antibodies were not able to detect the fusion protein. Even there may be other possibilities like the HA tag could have been cleaved off from the surface of a cell along with Ig κ, which acts as a leader sequence for target protein, thus leading to expression of only neo gene.