Working with mice

2.4.1. Mouse transgenes

LC-1

LC-1 is a transgene with direct control of luciferase and Cre recombinase expression under the promoter P_tetbi-1 (Schönig et al., 2002). This bidirectional promoter consists of seven tetO repeats flanked by two hCMV derived minimal promoters and is activated upon rtTA binding.

Lmx1b, floxed

In this transgenic mouse line, two loxP sites were introduced into introns downstream of exon 4 and upstream of exon 6 of the endogenous murine Lmx1b (Suleiman et al., 2007).

Cre recombinase expression results in an in-frame deletion of exons 4 – 6, containing the homeobox sequence. Mice were kindly provided by R. Johnson.

mTmG

The reporter construct mTmG comprises of membrane-targeted versions of tdTomato (mT) and an EGFP (mG) under the control of a chicken -actin core promoter with a CMV enhancer (Muzumdar et al., 2007). tdTomato and a downstream polyadenylation (pA) signal are flanked by loxP sites. This construct results in mT expression in all kinds of tissue under normal conditions. In case Cre recombinase is expressed, mT and the pA signal are cut-out and expression of mG starts. This construct enables to mark specific cell types with green fluorophores by specific promoter controlled Cre expression. Mice were kindly provided by T. B. Huber.

P2.5 Cre

The expression cassette for P1 bacteriophage Cre recombinase is regulated by a 2.5 kbp promoter fragment of the human NPHS2 gene (Moeller et al., 2003) encoding the protein podocin. The NPHS2 promoter is exclusively active in podocytes. The Cre recombinase excises flanked by loxP (floxed) gene sequences and for this reason, podocyte-specific gene deletions can be obtained.

P2.5 rtTA

The rtTA gene (reverse tetracycline-controlled transcriptional transactivator) is derived and modified from the E. coli tetracycline-resistance operon. The encoded protein is able to bind to tetO and activate transcription only if tetracyclines (e.g. doxycycline) are present.

The gene was put under the regulatory control of a 2.5 kbp fragment of the human NPHS2 promoter (Shigehara et al., 2003).

SM-CreER^T2(ki)

A construct encoding for the CreER^T2, an SV40 polyadenylation signal and a neomycin-resistance gene was integrated into the Sm22 locus resulting in a Sm22 promoter driven CreER^T2 expression while transgelin expression is abolished (Kühbandner et al., 2000).

CreER^T2 is a Cre recombinase fused to a mutant estrogen ligand binding domain, resulting in an inactive protein in the absence of tamoxifen. In this work this mouse line was used as a knock-out mouse model, the CreER^T2 protein was never activated with tamoxifen.

2.4.2. General handling and breeding

The mice are bred in euro standard type II and III cages in a conventional animal laboratory of the University of Regensburg. Offspring was marked by footpad tattoos and tail biopsied 4 – 21 days after birth and separated from the mother at 19 – 28 days of age. Animals had unlimited access to drinking water and complete food in a 12 h day/night cycle.

2.4.3. Genotyping

DNA isolation

Mouse biopsies were digested with 0.2 g/L proteinase K in 700 µL tail buffer overnight in a hybridization oven at 50 °C under rotation. The next day samples were vortexed and centrifuged (10 000 g, 30 min, 4 °C) to remove remaining insoluble material. Thereafter, 600 µL isopropanol was added to the supernatant in a fresh micro vial and it was vigorously inverted to precipitate DNA. After centrifugation (10 000 g, 30 min, 4 °C) supernatant was sucked off and 70% ethanol (500 µL) was added. The vial was inverted several times and centrifuged (10 000 g, 15 min, 4 °C) again. Finally, ethanol was discarded, DNA was completely dried at 40 °C for at least 30 min and resolved in 30 - 200 µL TE buffer overnight in a hybridization oven at 50 °C.

PCR

Mouse genotyping for all six transgenes was realized by PCR. In all cases, 0.5 µL DNA was diluted with DNA free water to 20 µL in a 0.2 mL micro tube. Additionally, a positive control of a known sample and a water control were also prepared. The master mix containing the appropriate primers was freshly prepared according to Table 2.3 and 5 µL was added to every sample. Immediately after a quick mix samples were put into a thermal cycler and the PCR program was started (Table 2.4 and Table 2.5). After the program finished samples were kept at 4 – 12 °C. A complete list of the primers can be found in chapter 2.1.6.

Table 2.3: Master mixes for genotyping mouse transgenes Cre, Lmx1b floxed, mTmG, P2.5 Cre, rtTA, and Sm22 (KO).

Ingredients Volume for all transgenes

except P2.5 Cre

Volume for transgene P2.5 Cre

10x Thermopol buffer 2.5 µL 2.5 µL

DMSO / 0.75 µL

Primer (100 µM), each 0.15 µL 0.1 µL

dNTPs (10 mM) 0.5 µL 1.0 µL

Taq polymerase 0.25 µL 0.25 µL

H2O ad 5 µL ad 5 µL

Table 2.4: PCR protocols for all transgenes except P2.5 Cre

Step T t Comments

Melting DNA 94 °C 5 min

Melting amplicon 94 °C 30 s

35 cycles

Annealing variable 1 min 58 °C: Lmx1b floxed, rtTA

61 °C: mTmG, Cre

63 °C: Sm22 (WT), Sm22 (KO)

Elongation 72 °C variable 45 s: Lmx1b floxed, rtTA

60 s: mTmG, Cre

20 s: Sm22 (WT), Sm22 (KO) Final elongation 72 °C 5 min

Table 2.5: PCR protocol for P2.5 Cre

Step T t Comments

Melting DNA 94 °C 3 min

Melting amplicon 94 °C 45 s

30 cycles

Annealing 55 °C 45 s

Elongation 72 °C 2 min

Final elongation 72 °C 10 min

Agarose gel electrophoresis

5x DNA loading buffer was added to PCR samples and mixed well. In case the desired PCR amplicon was < 300 bp, xylene cyanol FF was used as a dye, for amplicons > 300 bp bromophenol blue. A 2% agarose gel was made by solving 3 g agarose in 150 mL 1x TAE buffer by heating in a microwave oven. For DNA visualization the intercalation dye ethidium bromide (60 µL/gel) was added. The liquid was cast in the apparatus, the combs were added and remaining air bubbles removed. After approx. 30 min of cooling at RT gel became solid and 12 µL PCR product with loading buffer was added per well. The 2-log DNA ladder was used for size estimation. Electrophoresis was conducted in 1x TAE buffer at a constant voltage of 150 V. DNA bands were visualized and documented by ultraviolet illumination in the GelDoc^TM system.

2.4.4. Collection and analysis of urine samples

Collection of urine

Urine samples of animals younger than 21 days and samples taken on the day of mouse perfusion were spontaneously voided urine samples (spot urine). At all other occasions, urine was taken over 24 h by keeping animals separately in a specialized cage for one day.

Spot urine samples from animals tested for survival were taken (if possible) on day 4, 10 and 21 after birth. Additionally, 24 h urine was taken at the age of 6 and 13 weeks. Spot urine at the day of perfusion was taken from all animals regardless of experimental series.

Qualitative and quantitative urine analysis

For qualitative determination of urinary proteins, SDS-PAGE with 1 µL spot urine or a 1:500 volume dilution in water of 24 h urine was conducted. For a rough estimation of urine albumin content, different BSA amounts (1, 3, 10 and 30 µg) were loaded on the first four lanes. The protocol for SDS-PAGE is described in chapter 2.3.2.

The quantitative protein content was measured using the Bradford test with a sample dilution of 1:20, as described in chapter 2.3.1.

The urine creatinine mass concentration was determined by the Jaffe reaction. Because of urine volume limitations, urine creatinine was measured only once with 5 µL/well in a 96-well plate. Urine was diluted to 50 µL with water (1:10), whereas water blank (50 µL) and a creatinine standard curve (30, 15, 7.5 and 3.75 mg/L) were pipetted in doublets. 150 µL of the master mix (Table 2.6) was added in fast succession. After 10 min reaction time, the colorimetric product was measured spectrophotometrically at 540 nm. To calculate the mass concentration of samples, linear regression of the standard curve was calculated and mass concentration was obtained using equation (3).

𝛽(𝑐𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒) = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ×𝑐𝑜𝑟𝑟. 𝐴₅₂₀× 𝑦 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

𝑠𝑙𝑜𝑝𝑒 (3)

Table 2.6: Master mix for Jaffe reaction for one urine sample

Volume Ingredients

50 µL 50 µL 50 µL

1.2 M 35 mM 1.6 M

Trichloroacetic acid Picric acid

Sodium hydroxide solution

2.4.5. Induction of Lmx1b knock-out

The inducible podocyte-specific Lmx1b knock-out mouse line received 2 g/L doxycycline in the drinking water to induce Cre expression. Normal drinking water was replaced by induction solution containing doxycycline and sucrose in the morning 7 days before animal perfusion. The solution was freshly prepared every two days and administered in lightproof bottles in excess.