Generation of Bcl11b Mutant Mice

A DNA fragment harboring C to A nucleotide change that generates Bcl11b^N797K protein was generated by overlap PCR, and replaced with wt exon 4 in the target vector, which was used to generate the Bcl11b^flox allele (9 (link)). The targeting vector for Bcl11b^N797K mutation was transfected into M1 embryonic stem (ES) cells as previously described (9 (link)), and ES clones which underwent homologous recombination were identified by PCR with an appropriate primer set. Bcl11b^+/N797K mice were generated by crossing C57BL/6NJcl mice purchased from CLEA Japan (Shizuoka, Japan) with chimeric mice generated from ES clones with Bcl11b^+/N797K genotype. The Bcl11b<σπ>Δ</σπ> allele was generated from the Bcl11b^fl allele (9 (link)) by crossing Bcl11b^+/fl mice with EIIa-Cre transgenic mice that were purchased from Jackson laboratory (#003314). Cd4^ΔS mice was previously described (24 (link)). CD45.1 congenic mice and Rag1-deficient mice were obtained from Jackson Laboratories. All mice were maintained in the animal facility at the RIKEN IMS. All animal procedures were in accordance with institutional guidelines for animal care and the protocol (2020–026) approved by the Institutional Animal Care and Use Committee of RIKEN Yokohama Branch. All the mice were sacrificed by euthanasia with CO₂ overdose following anesthesia using isoflurane.