ES cells transfection and screening

Generated targeting constructs were transfected into ES cells following the procedure summarized in Figure 5.20. To improve the electroporation efficiency, various protocols were followed and are described below. Results are represented in Table 5.4.

Figure 5.20 Different stages of ES cells transfection and screening.

Generated targeting construct was linearized and electroporated into ES cells, which were previously expanded on EF plates. After electroporation and selection (positive and negative selection), ES cell clones were picked into 24-well plates and expanded. Once ES cells reached confluency, they were split into an A-plate (planned for freezing) and a B-plate (used for screening).

5.4.3.1. Transfection and screening of targeting construct I

The cloning procedure of targeting construct I, TOPO-[KA-GFP-Neo-LA]-TK, was described in section 4.5.1.1, and its map is schematically depicted in Figure 5.21.

The transfection of targeting construct I into ES cells was performed as follows (Prof. Dr. Thomas Hehlgans, Institut of Immunology Regensburg, personal communication):

ES cells (kindly provided by Prof. Dr. Klaus Pfeffer) were seeded on a monolayer culture of EF cells. EF CD1 cells were kindly provided by the lab of Prof. Dr. Thomas Hehlgans, and

were expanded for three weeks. Prior to electroporation, their mitosis was inactivated by incubation with Mitomycin C.

The electroporation of 20 µg linearized DNA into 5x10⁶ ES cells/cuvette was performed with Biorad electroporator (340 V, 250 µF, constant time between 5.2 and 6.4 ms). After 10 days selection (with Neomycin and Gancyclovir), 200 ES clones were picked and cultured for two weeks in ES cell medium without any selection pressure. Afterwards, ES cells plates were frozen and genomic DNA was phenol/chloroform extracted from each clone separately (see section 4.5.7.1). The screening of ES clones was performed by using a 5’ and 3’ DIG-labeled Southern probe. From 200 clones, only one was screened positive for homologous recombination. Unfortunately, because the whole procedure was very much time consuming (3 months), the corresponding cryo-conserved ES clone did not survive after thawing the A-plate.

Figure 5.21 Schematic representation of MMD targeting construct I.

TOPO[KA-GFP-Neo-LA]-TK targeting vector contains a reporter cassette (GFP, in green) and a positive selection cassette (Neo^R, inpink) with loxP sites (black arrows). Both cassettes are flanked by a homologous short ( 0.5 kb) and long (2.5 kb) arms displayed as bold lines also on the endogenous locus. A negative selection cassette (TK, in grey) was inserted outside the homologous regions. After homologous recombination (dotted lines), part of exon II and the following intron (regular line) were replaced with Neo^R and GFP cassettes to generate the targeted locus. Southern blot analysis was performed after digestion of the genomic DNA with XbaI restriction enzyme. Detection followed by using a 5’ and 3’ probes (green lines). The expected size of the WT band is 4.9 kb and after recombination the 5’probe detects a 2.1 kb and the 3’probe a 3.3 kb band (dashed arrows). (B) BamHI;

(E) EcoRI; (H) HindIII; (K) KpnI; (N) NotI and (S) SalI restriction sites.

5.4.3.2. Transfection and screening of targeting construct II

The recombination efficiency obtained with targeting vector I was not sufficient (Table 5.4).

Possibly the length of the homology regions may have influenced the homologous recombination efficiency (Hasty et al., 1991), therefore in the second targeting construct the short 500-bp arm (KA) was replaced by a longer 4600-bp arm (see section 4.5.1.2). The map of targeting construct II is represented in Figure 5.22.

Figure 5.22 Schematic representation of MMD targeting construct II.

TOPO-[LLA-GFP-Neo-La]-TK targeting construct includes GFP, Neo and TK cassettes. The homologous regions (in bold) consist of the long (4.6 kb) and short (2.5 kb) arms. The Southern blot analysis was performed following digestion of the genomic DNA with XbaI, and screening with the 3’probe (green line). The expected WT band has the size of 4.9 kb and after homologous recombination an additional 3.3 kb band was expected. (B) BamHI; (E) EcoRI; (H) HindIII; (K) KpnI;

(N) NotI and (S) SalI restriction sites.

Amplification and purification of large amounts of this construct were possible only after growing the transformed bacterial DH10B strain under non-stringent conditions (50 µg/ml ampicillin and at 32°C). Afterwards, the targeting construct II was introduced into ES cells following the protocol described in section 5.4.3.1. Additionally, an Amaxa electroporator was used to electroporate 2.5x10⁶ ES cells with 10 µg DNA following the manufacturer’s instructions. Although no negative selection was done, only 60 Neo^R clones were picked and screened negative for homologous recombination. The absence of homologous recombination and a high mortality rate of the electroporated ES cells were the two main reasons to design a third targeting construct.

5.4.3.3. Transfection and screening of targeting construct III

Taking advantage from the results of previous targeting vectors, targeting construct III, pBS-[SA3-GFP-Neo-LA]-DT, was generated as described in section 4.5.1.3 (Figure 5.23).

Figure 5.23 Schematic representation of MMD targeting construct III.

The pBS-[SA3-GFP-Neo-LA]-DT vector contains in addition to GFP and Neo cassette, a DT negative selection cassette (in blue). The bold lines depict the homologous short (1.5 kb) and long (2.5 kb) arms. The WT genomic DNA was digested either with NcoI or XbaI to generate a 4.3 kb or a 4.9 kb band, respectively. After homologous recombination (dotted lines), an additional band with the size of 5.7 kb by using the 5’probe, and of 3.3 kb by using the 3’probe is expected. (B) BamHI; (E) EcoRI;

(H) HindIII; (K) KpnI; (N) NotI, and (S) SalI restriction sites.

pBS-[SA3-GFP-Neo-LA]-DT was electroporated into a new batch of ES cells which were kindly provided by Dr. Marina Karaghiosoff (Institut of animal breeding and genetic, Vienna, Austria). In addition, a new FCS batch was tested for these ES cells. To improve the plating and adherence of electroporated ES cells, a new batch of EF cells was prepared from inbred mice expressing the neo^r gene (see section 4.5.2.3). EFneo^r cells have the advantage to preserve the non-differentiated morphology of ES cells during their selection more efficiently.

In addition, EFneo^r cells were inactivated via radiation before cryo-conservation. Whenever needed, they were thawed and directly used without expansion. In this way the targeting experiment time was reduced to at least 3 weeks.

For electroporating the targeting construct III, the Amaxa protocol described in section 5.4.3.2 was followed. Unfortunately only 29 Neo^R clones were picked from six electroporation reactions. In contrast, Biorad electroporation of 50 µg linearized DNA into 3x10⁷ ES cells (500 V, 340 µF and between 7.1 and 8.3 ms) allowed to pick 600 Neo^R clones from five electroporation reactions. Again all clones were screened negative for homologous recombination.

5.4.3.4. Transfection and screening of targeting constructs IV

Suspecting that the genomic region between exon I and II could be inappropriate for a homologous recombination event in construct IV, the short homologous arm (1.4 kb) was positioned between exons IV and V, and the long arm (4.4 kb) upstream of exon VI.

Consequently, after homologous recombination exons V and VI are replaced with the Neo cassette, which disturbs the ORF of the gene. Although, the first four exons of mMMD will be translated, however the protein will be lacking 122 amino acids (out of 238) which may severely affect its membrane integration and therefore its functional property.

Figure 5.24 Schematic representation of the MMD targeting construct IV.

pBS-[SAex5-Neo-LAex6]-DT targeting construct contains Neo and DT cassettes. The homologous regions (in bold) consist of a short (1.4 kb) and a long (4.4 kb) arms. After homologous recombination (dotted lines), exons V and VI were replaced with the Neo cassette. A 3’southern probe (green line) was used to distinguish between a WT (6.2 kb) and knock-out (8.6 kb) band.

The targeting construct IV, pBS-[SAex5-Neo-LAex6]-DT (Figure 5.24), was cloned as described in section 4.5.1.4. It was electroporated following the instructions described in section 5.4.3.3. This led to the isolation of 551 Neo^R clones. Genomic DNA was isolated following a fast method (see section 4.5.7.1) which reduced enormously the hands-on time needed in phenol/chloroform genomic DNA extraction. The Southern blot screening was performed using radioactively labeled probes, reducing the screening procedure to 2 weeks.

Again, all 551 Neo^R clones were negative for homologous recombination.

5.4.3.5. Transfection and screening of targeting vector V

The elements of targeting vector V are the same as described in Figure 5.21. However, because no enrichment of clones with a successful homologous recombination event was seen with the previously described targeting vectors, negative selection markers were omitted from construct V. This has an additional advantage, which is to reduce the size of the vector.

TOPO-[KA-GFP-Neo-LA] was chosen to electroporate ES cells because it was the only vector, which yielded to a single homologous recombination event. After isolation and expansion of 553 Neo^R clones, the genomic DNA was extracted and Southern blot analysis was carried out with radioactively labeled probes. Besides the expected wild type (WT) band, many clones displayed additional bands, which however can not be explained by a homologous recombination event. It seems that the targeting construct integrated inappropriately into the MMD locus.

Table 5.4 Summary of the targeting experiments

Targeting constructs Number of isolated clones Number of positive clones TOPO-[KA-GFP-Neo-LA]-TK 200 1

TOPO-[LLA-GFP-Neo-LA]-TK 60 0 pBS-[SA3-GFP-Neo-LA]-DT 629 0 pBS-[SAex5-Neo-LAex6]-DT 551 0 TOPO-[KA-GFP-Neo-LA] 553 0

5.5. Mouse MMD silencing in NIH3T3 and RAW264.7 cell