PiggyBacHR Genome Eng

Vector map

Preparation of Homology Arms

5’ Homology Arm (5’HA)
1. PCR the 5’ HA and add any restriction site you choose from the 5’ MCS except BsaI to the forward primer. Add a BsaI site and the sequence GTTAA to the 3’ end of the reverse HR primer. Homology arms should be between 1 and 0.5 Kb. The 5’ HA cannot contain a BsaI site.

5’ HA forward primer sequence

Restriction Site in 5’MCS of PBHR vector

5’gccgccXXXXXX--------------------3’

extra Sequence    sequence of your 5’HA (~20 bp)

5’ HA reverse primer sequence

            BsaI site      PiggyBac TTAA

5’ gaagaaGGTCTCgTTAA-------------3’

extra Sequence              sequence of your 5’HA (~20 bp)

2. Run out the PCR product on an agarose gel.
3. Cut of the band of the correct size and isolate the fragment.
4. Cut the fragment with the 5’MCS enzyme you chose and with BsaI.
5. Clean up the reaction and quantify the DNA.

3’ Homology Arm (3’HA)

1. PCR the 3’HA and add the BspQI site to the forward primer. Add any site you wish from the 3’ MCS except BspQI to the reverse primer. Homology arms should be between 1 and 0.5 Kb. The 5’ HA cannot contain a BspQI site.

3’HR forward primer sequence

BspQI Site Sequence of your 3’HA (~20 bp)

5’taataaGCTCTTCTTAA------------------------------3’

extra sequence    PiggyBac TTAA

3’HR Reverse primer sequence

Restriction site from the 3’ MCS

ttcttcXXXXXX------------------------------------

extra sequence    sequence of your 3’HA(~20bp)

2. Run out the PCR product on an agarose gel.
3. Cut of the band of the correct size and isolate the fragment.
4. Cut the fragment with the 3’ MCS enzyme you chose and with BspQI.
5. Clean up the reaction and quantify the DNA.

Preparation of PiggyBac Homologous Recombination Vector

Determine the order that the homology arms will be cloned into the PBHR vector based on which restriction sites are used.

1. Cut the PBHR vector with the 5’ MCS enzyme you chose and with BsaI.
2. Run out the reaction on an agarose gel.
3. Cut out the band of the correct size and purify.
4. Quantitate the DNA.
5. Ligate in the 5’HA fragment. Use a recombination deficient competent bacterial strain for the transformation.
6. Screen colonies for the clone of interest.
7. Cut the PBHR vector containing the 5’HA with the enzyme you chose in the 3’ MCS and with BspQI.
8. Run out the reaction on an agarose gel.
9. Cut out the band of the correct size and purify.
10. Quantitate the DNA.
11. Ligate in the 3’HA fragment. Use a recombination deficient competent bacterial strain for the transformation.
12. Screen colonies for the clone of interest.

Gene Correction using the PiggyBac Homologous Recombination Vector

1. Cotransfect the cells containing the mutation with the TALENs and the PBHR vector.
2. 24 hours later split the cells.
3. 24 hours later switch to media containing puromycin.
4. Continue growing cells in puromycin until distinct colonies begin to form.
5. When large enough, pick colonies to individual well of a 6-well plate under puro selection.
6. Isolate genomic DNA from each line and screen by PCR for clones that have the PBHR vector integrated at the correct location. Design one genomic primer outside each homology arm and on in the PBHR vector.
7. Transfect lines with correct integrations with the piggyBac transposase.
8. 48 hours later split the cells
9. 24 hours later switch to media containing ganciclovir.
10. Continue growing cells in ganciclovir media until distinct colonies begin to form.
11. When large enough, pick colonies to individual well of a 6-well plate under ganciclovir selection.
12. Colonies should now be GFP-, puros and ganciclovirr.
13. Isolate genomic DNA from cell lines and use PCR to confirm the absence of the PBHR vector and to amplify the region containing the correction.
14. Confirm correction by DNA sequencing.