Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

The overall aim of this protocol is to efficiently target transgenes to loci of interest in human embryonic stem cells using custom designed zinc finger nucleases to do this. Human embryonic stem cells or HSCs are expanded to generate enough material for electroporation, then digested to yield single cells. Next mRNA encoding two custom design zinc finger nucleases is co electroporated into the HSCs with the linearized EGFP donor sequence and cells are then plated onto fresh mouse embryonic, fibroblasts or MEFs.

The electroporated zinc finger nucleases Introduce a double strand break into the first exon of the pit X three locus in the cells. Then the EFGP donor sequence, which is flanked by targeting arms with homology to the DNA doublet strand Brake site sequence is incorporated during cell division after 14 to 21 days. One third of an individual HESC colony is passaged onto each well of a matrigel coated 96 well plate and the other two thirds is transferred to a corresponding well of a 48 well plate containing fresh ME Fs.When the cells on the 96 well plates reach co fluency, they're screened for incorporation of the donor sequence by PCR.

Southern blotting is then performed to confirm correct integration. Once positive clones are confirmed, they are expanded and then frozen for further use. The main advantage of this technique over existing methodology, such as conventional homologous recombination, is that zinc fingers create predetermined DNA double strand breaks, which facilitate the efficient targeting of transgenes to predetermine loci in human embryonic stem cells.

Although here we introduce a trans gene to the pit X three locus of human stem cells, this method can be adapted to any other system where the use of reporter cell lines would be advantageous. Begin this protocol by expanding human embryonic stem cells or HSCs for transfection as described in the accompanying document. When the colonies become 60 to 70%confluent, place the tissue culture plate on a dissecting microscope.

Use a 21 gauge needle on the end of a two mil liter pipette to groom the colonies by removing differentiated colonies or colony cores. Next to wash the HESC colonies aspirate the medium. Then add five milliliters of HBSS and incubate for two minutes.

After the incubation aspirate, the HBSS add five milliliters of Accutane and incubate for 10 to 20 minutes at 37 degrees Celsius and 5%CO2 until the colonies are rendered single cells as viewed under a microscope. Using a one milliliter pipette tri the cells. Next, add 10 milliliters of HESC medium and using a 40 micron cell strainer, filter it into a 15 milliliter conical tube.

To remove any clumps of cells, centrifuge the flow through at 160 times gravity for five minutes. After the spin Resus, suspend the cell pellet in five milliliters of fresh HESC medium and spin again To remove any residual Accutane solution. Resuspend the cells in HESC medium containing 10 micromolar Y 2 7 6 3 2 and transfer them to 100 millimeter dish pre-coated with gelatin incubated 37 degrees Celsius, 5%CO2 for a minimum of 30 minutes.

During this time, the MEFs will adhere to the gelatin coated dish. After incubation, use a one milliliter pipette to collect the non-adherent HE SSCs into a fresh 15 milliliter centrifuge tube and determine the cell density using a hemo cytometer in preparation for electroporation. Transfer 7.5 million HSCs as prepared in the previous section of this video to a new 15 milliliter conical tube and centrifuge at 160 times gravity for five minutes.

Following the spin Resus, suspend the cell pellet in 0.8 milliliters of ice cold 0.22 micron filtered HBSS transfer the solution to a sterile 0.4 centimeter electroporation vet. Next, add 30 micrograms of TVH pit X three forward targeting. Construct DNA and mix gently with a 200 microliter pipette incubate on ice for five minutes during the incubation thaw and aliquot of pit X three ZFN mRNA on ice.

Once thawed transfer 7.5 microliters to the DNA cell mixture into the electroporation Q vet mixed gently with a 200 microliter pipette. Press the red button on the electro to transfect the cells. Then using a 200 microliter pipette transfer the contents from the Electroporation Q Vet to a 15 milliliter tube containing 10 milliliters of HESC medium.

Wash the Q VET twice with 200 microliters of HESC medium and collect the wash solutions in the same 15 milliliter tube. To remove the resulting mucus like debris consisting of DNA and protein from the lysed cells. Centrifuge at 160 times gravity for five minutes.

After the spin, aspirate the supernatant and gently tap the cell pellet to loosen Reese spend in 30 milliliters. M-E-F-C-M supplemented with RH FGF two and 10 micromolar Y 2 7 6 3 2 and plate onto three 100 millimeter dishes containing MEFs. Incubate at 37 degrees Celsius 5%CO2 95%O2.

Changing the medium daily on day two, withdraw Y 2 76 32 and gross cells in HESC medium only. After two to three days, the HES CS should be 50 to 70%confluent at this point. Begin antibiotic selection.

Maintain selection by adding fresh HESC per mycin medium daily. After about seven days, small colonies should be visible if antibiotic resistant MEFs are not used. MEFs can be supplemented by adding an additional one times 10 of the fourth cells per square centimeter.

14 to 21 days following selection when colonies have reached about one millimeter in diameter. Prepare plates for expansion and screening by adding 75 microliters of matrigel or gel TRE to each well of 3 96. Well plates also prepare 3 48 well plates of MEFs at two times 10 to the fourth cells per square centimeter incubate both sets of plates overnight at 37 degrees Celsius, 5%CO2 the following day.

Use a 21 gauge needle on the end of a two milliliter pipette to dissect HESC colonies into 16 to 25 pieces by cutting a grid. Next, aspirate the matrigel from 96 well plates and replace it with CM MEF medium supplemented with R hf, GF two and per mycin. Wash the MEFs in the 48 well plates with HBSS and then add HESC medium with per mycin using a 200 microliter pipette.

Gently transfer one third of the dissected colony into a 48 well plate for expansion. Transfer the other two thirds of the dissected colony into the corresponding 90 well plate containing supplemented C-M-M-E-F medium. The most difficult aspect of this procedure is the transfer of colony pieces.

We suggest that you practice the transfer and dissection color pieces beforehand. Maintain both plates by changing the antibiotic supplemented media daily. Expand until the 96 well plates are 100%confluent once the plates are confluent.

Remove all medium from the plates by inverting, then blot onto paper towels. Proceed to prepare the genomic DNA for PCR as described in the accompanying document after Resus suspending the DNA in TE or TO 0.1 E.Perform PCR using the expand long template PCR system as described in the accompanying document, use one to two microliters of resuspended DNA per 10 microliters PCR reaction following PCR screen for positive clones by electrophoresis and confirm by southern blotting. Once positive clones are confirmed, proceed with subculture and freeze the remainder of the colony fragments to engineer a PIT X three.

Reporter cell line hecs. Were co-ed with a custom designed pit X three zinc finger pair and the pit X three EGFB specific DNA donor vector as described in this video article following PERY and selection, positive HESC clones were detected via genomic PCR screening as can be seen here. There were numerous positive clones as indicated by bands at 9 84 base pairs.

Southern blot hybridization of these PCR positive clones was then performed using five and three specific probes. This illustration shows the schematic of the linearized targeting vector as well as the location of the five and three external probes here. The five and three southern blots of both wild type and targeted alleles are shown.

The single band in the wild type lane indicates two identical alleles following correct targeting of the transgene to the pit x three locus two bands are observed. One corresponding to the correctly targeted allele the other to the wild type allele. Once mastered, this methodology can be performed in two to three months.

After watching this video, you should have a good understanding of how to target human stem cells using zinc finger nucleases, as well as how to use PCR and southern blot strategies to screen for correctly targeted cls.