Figure 3.

Generation of germ cell and Sertoli cell-specific Lifr-KO mice. (A) Schematic of the conditional Lifr allele detailing the location of PCR primers designed to detect Cre-mediated recombination and the expected sizes of the PCR products. Germ cell and Sertoli cell Lifr-knockout mice were generated as described in materials and methods. Representative PCR analysis of genomic DNA isolated from the testes of heterozygote Stra8-Cre^−/−;Lifr^wt/tm1c and Stra8-Cre^+/−;Lifr^wt/tm1c (B) and Amh-Cre^−/−;Lifr^wt/tm1c and Amh-Cre^+/−;Lifr^wt/tm1c (C) mice confirmed recombination of the conditional Lifr allele upon Cre exposure. Representative PCR analysis of testicular cDNA from Stra8-Cre^−/−;Lifr^wt/tm1c and Stra8-Cre^+/−;Lifr^wt/tm1c (D) and Amh-Cre^−/−;Lifr^wt/tm1c and Amh-Cre^+/−;Lifr^wt/tm1c (E) mice confirmed Lifr mRNA is expressed in germ cells and Sertoli cells, evidenced by the presence of a mutant transcript following Cre-mediated gDNA recombination. Representative Western blot analysis of whole testis protein extracts from Stra8-Cre^−/−;Lifr^tm1c/tm1c (WT) and Stra8-Cre^+/−;Lifr^tm1c/tm1c (GC-KO) (F) and Amh-Cre^−/−;Lifr^tm1c/tm1c (WT) and Amh-Cre^+/−;Lifr^tm1c/tm1c (SC-KO) (G) mice. A significant reduction in testicular LIFR protein is observed only when Lifr is disrupted in Sertoli cells (SC-KO; unpaired t-test; p = 0.001). For GC-KO and SC-KO blots, both LIFR and TUBA bands have been cropped from single gel blot images which are included in the supplementary materials with the cropped areas highlighted. Tubulin-alpha (TUBA) was used as a loading control. Values are expressed as mean ± S.E.M of n = 7 samples per genotype.