Screening of embryonic stem cell colonies and microinjection

After electroporation of the targeting construct, many ES cell colonies gained resistance against geneticin but only few of these colonies had actually undergone

homologous recombination. Southern hybridization and PCR dependent strategies have been used for screening of homologous recombination events in selected ES cell colonies. Both procedures were essential as southern hybridization could test the correct recombination event only at 3’ side and PCR screening could test only the 5’

side of the locus (see Figure 2.8).

2.2.13.1. Southern based screening

For Southern hybridization based screening, it is essential to select an external probe whose sequence matches with the genomic sequence but not with the targeting vector sequence. Initially we selected an external probe from the 5’ side of the targeting vector. However, this probe recognized a DNA smear rather than a single band (data not shown). Closer inspection of the probe sequence by BLAST searches in mouse genome DNA database showed that this probe contained sequence elements that were repeated widely in the genome with sequence conservation approaching 90%. Therefore, we thrusted this probe aside and selected another external probe from the 3’ side of the targeting vector (Figure 2.8). This probe was amplified using primers Pr-18 and Pr-19 and it recognizes a single DNA band of 15.5 kb size from HinDIII digested WT genomic DNA (Figure 2.9).

Figure 2.9 Testing of the external probe. Different quantities of genomic DNA from WT embryonic stem cells was restriction digested with HinDIII enzyme, separated by gel electrophoresis and Southern blotted. Upon hybridization with the external probe amplified using P-18/P-19 primers, a single DNA fragment of 15.5 kb size was detected as expected.

Upon homologous recombination, a HinDIII restriction site is introduced at 3’ side of the neomycin cassette. This would result in recognition of a new 9.2 kb fragment

after Southern hybridization in addition to the 15.5 kb fragment coming from the WT locus. Screening of ES cell genomic DNA obtained from the first 23 clones (plate F) using this procedure resulted in identification of 3 positive clones (Figure 2.10).

However, one of these clones, F13, was negative according to PCR based screening. Probably, the 3’ side of the targeting vector was inserted into the genome by homologous recombination of the longer arm and 5’ side was randomly inserted.

After PCR based screening procedure was established, the rest of the ES cell colonies were initially screened with PCR, the positive ones were selected and retested by southern hybridization.

Figure 2.10 Southern screening of ES cell colonies. Genomic DNA samples from the selected colonies were screened by Southern hybridization. F11 and F13 clones have a second band of 9.2 kb as a result of recombination.

2.2.13.2. PCR based screening

An alternative and easier strategy for screening for positive clones was performed by PCR. In this approach, two primers were selected: one from the neomycin cassette sequence and the other from the genomic sequence that was not included in the targeting vector (Figure 2.8). 5’ side of the targeting vector was chosen for PCR screening as it was shorter. Two 5’ side primers (up1 and 2) and two 3’ side primers ( low1 and 2) have been selected and used as primer pairs (4 pairs). Up1-Low2 primer pair was chosen for further screening as it yielded the best PCR product tested on genomic DNA samples with known genotype according to Southern hybridization.

The other primer pairs also yielded the expected size fragments. The following PCR program was used for screening of all ES cell colonies. Taq polymerase was the

enzyme of choice as it yielded better results. After 25 cycles of PCR, additional Taq polymerase was added and 15 more cycles were performed. A 1910bp DNA fragment was amplified only from colonies that underwent homologous recombination at the 5’ arm (Figure 2.11).

ESC screen PCR cycle

95°C 10 min Initial denaturation 95°C 30 sec Denaturation 60°C 45 sec Annealing 72°C 3 min Extension 72°C 5min Final extension

4°C ∞ Storage

As PCR is a contamination sensitive procedure and the risk of contamination of negative colonies with cells from positive colonies is high during the colony picking procedure, the PCR results were not regarded as entirely trustable and the colonies were further checked by Southern hybridization. As a result of screening by PCR and Southern hybridization 4 positive clones have been identified out of 149 picked up colonies: F9, F11, A15 and C15.

Figure 2.11 PCR based screening of ES cell colonies. 50ng of genomic DNA from the indicated ES cell colonies was used as a template for PCR amplification. A 1.9 kb fragment is amplified only from clone F9, which was tested to be positive before by Southern hybridization (data not shown).

2.2.13.3. Microinjection of positive ES cell colonies and generation of chimera Frozen vials of A15 and F11 clones were thawed and expanded for 5 days on PMEF covered, recombinant LIF (ESGRO) supplied culture dishes. No signs of differentiation were eminent during the culturing period. The identity of both clones was reconfirmed via southern blotting and PCR before microinjection. Aliquots of ES

25X+15X

cells were taken and stored in liquid N2 for possible future microinjections. ES cells from clones F11 and A15 were microinjected into blastocyts from C57Black6/N mice by Monika Schindler (Animal Facility of MPI Experimental Medicine, Göttingen) and these chimeric blastocyts were implanted to pseudopregnant mice. The microinjection dates were 20/21 January 2003 respectively. We have obtained 4 male chimeras from clone A15 (7^th of February 2003) while clone F11 did not yield chimeric animals. The degree of chimerism was estimated to be between 30% and 90%. One of the four chimeras was eaten by his mother before reaching maturity.

The remaining 3 chimeras were transported to animal facility of Biochemistry 2. They were mated with C57Black6 female mice for the establishment of a mouse colony.

A second round of microinjection was performed on 19^th and 20^th of February 2003.

ES cells from clone F11 (refrozen vial) and C15 were expanded in the same way.

Out of these microinjections we obtained 9 chimeras on 9 / 10 March 2003 (8 from F11 and 1 from C15). Some of these animals have also been used in generation of stonin2 colony.