Deconstruyendo la categoría género en la escuela

3.5.1 Solutions

- Cystein: 2% L-Cystein in 0.1xMBS (pH7.8 at 23°C, adjusted with 5M NaOH). - Human Chorionic Gonadotropin (HCG): 1000 I.U./ml HCG in ddH2O.

- MEMFA: 0.1M 3-(N-Morpholino)-propanesulfonic acid (MOPS), 2mM EGTA, 1mM MgSO4; 3.7% formaldehyde (pH 7.4 at 23°C).

- 1xModified Barth’s Saline (MBS): 5mM HEPES, 88mM NaCl, 1mM KCl, 0.7mM CaCl2, 1mM MgSO4, 2.5mM NaHCO3(pH 7.6 at 23°C). Add the CaCl2 before use.

- MBS/high salt: 1xMBS with 50mM NaCl

- 0.1xMBS/Gentamycin: 0.1xMBS, 10g/ml Gentamycin - 0.5xMBS/BSA: 0.5xMBS, 1mg/ml BSA, 10g/ml Gentamycin

- MBS/CS: 0.8xMBS high salt with 20% chicken serum, 200U Penicillin/ml, 200g/ml streptomycin stored at -20°C

3.5.2 Experimental model organism

Adult wild-type Xenopus laevis frogs (Xenopus Express) were used. The frogs were kept in charcoal filtered water with a temperature of 17-19°C with a population density of 5l water per frog. The animals were fed three times per week with Pondsticks Premium brittle (Interquell GmbH, Wehringen).

3.5.3 Ovulation stimulation

Female Xenopus laevis were injected 800 units of human chorionic gonadotropin (Sigma) into the dorsal lymph sac. After incubation at 18-20°C over night, females started to lay eggs 12-18 h later.

3.5.4 Isolation of testis

Male frogs were anaesthetized in 0.1% 3-Aminobenzoeacid-ethyl-ester in ddH2O for 30 min and cooled down in iced water. The male frogs were killed by decapitation. The testes were removed from the abdominal cavity and separated from the frog body, to which they are connected to and isolated from the abdominal cavity. Testes were stored in MBS/CS at 4°C for maximal 7 days.

3.5.5 In vitro fertilization

In-vitro fertilization was performed by homogenizing testis tissue in 1 x MBS and mingle it with freshly laid eggs. The fertilized eggs were cultured in Petri dishes with 0.1xMBS at 16-23°C until the stage was reached for injection procedure. Embryos, which served as uninjected control, were cultured in parallel.

3.5.6 Dejellying of fertilized eggs

The fertilized eggs are covered by a jelly coat, which was removed about 60 minutes after fertilization by a 2% cysteine solution pH 7.8 for with gentle agitation in an Erlmaier glass flask until the eggs were in direct contact with each other as a sign for total removal of the jelly coat. Embryos were washed three times with 0.1 x MBS and cultured in Petri dishes with 0.1xMBS at 16-23°C.

3.5.7 Preparation of microinjection and needle calibration

Injection needles were pulled from capillaries with the Microneedle Puller (setting: heat: 800; pull: 35; vel: 140; time: 139; Sutter Instrument, model P-87). The needle

was adjusted to the holder of the injection equipment (Medical System, model Pi- 100), and the tip of the injection needle was calibrated by cutting the tip at the point were the flexible part becomes more rigid with Dumont tweezers. Shortly before the injection, the needle was filled with 1-2 l nucleotide acid containing RNase free DEPC water.

3.5.8 Microinjection of Xenopus embryos

The injection volume was adjusted by choosing the injection pressure of 25-30 psi with an injection time of 30 ms-1 s. With each shot, 5nl nucleotide solution containing was injected into a blastomere of the two to eight cell stage embryos into the animal hemisphere. After injection, the embryos were transferred into new dishes covered with 1% agarose in 0.1 x MBS with Gentamycin and incubated in 0.1 x MBS at 16- 23°C until the desired developmental stages was reached. The saline was changed every day.

3.5.9 CHD4/Mi-2ß and Sip1 gain- and loss-of-function analysis

The gain of function of wtCHD4 or Sip1 was performed by microinjection of 1ng/5μl CHD4 or Sip1 mRNA in one blastomere of a two-cell stage Xenopus embryo respectively, as described in 3.5.8 to increase the CHD4 activity.

Microinjection of dnCHD4 mRNA on the other hand lead to the over expression of CHD4 protein with a functionally inactive ATPase domain, which diminishes the endogenous CHD4 protein ATPase function by competitive inhibition without disturbing the association with complex subunits or co-factors of the NuRD complex. For loss of function analysis we designed Morpholino oligonucleotides, complementary to the translation start side of endogenous CHD4 mRNA.

The injection of these antisense nucleotides at a concentration of 40ng was shown to inhibit the translation of endogenous CHD4 protein. To test the efficiency of CHD4 Morpholinos to reduce translation of CHD4 protein, we subcloned the first 363 amino acids of the CHD4 in-frame upstream of the 6x Myc-tag cassette of the pCS2+MT6 vector, with and without the 5’ untranslated region, which is complementary to the CHD4 Morpholino. The in-vitro translation of these test constructs was analysed with increasing amounts of CHD4 Morpholinos, followed by Western blot detection to evaluate the relative levels of the CHD4-Myc protein (Linder et al, 2007) (Figure 9).

The described concentrations have been determined to be the optimal amount for functional interference without generating toxic side effects. To distinguish between left and right, embryos were co-injected with GFP mRNA or Alexa Dextrane and pre- sorted before fixation. The modulation of neural gene expression by CHD4 mis- expression was analyzed by whole mount in situ-hybridization as described in 3.4.6. As controls, we compared the gene expression patterns and morphology of the injected side with the uninjected control side of the embryos, as well as with completely untouched wild-type embryos. Neither injection of gfp mRNA, nor the injection of 5% Alexa Dextrane in RNase free DEPC water led to phenotypic alterations of gene expression or morphology.