Construction of Cre-N-Intein-N and Intein-C-Cre-C fusion proteins
To generate the Cre-N-Intein-N and Intein-C-Cre-C fusion proteins, the N- (aa 19–59) and C-terminal (aa 60–343) fragment of Cre recombinase, were first PCR amplified. The N terminal fragment (Cre-N) is fused to the intein-N which contains 123 N-terminal amino acids of the intein sequence of DnaE split intein to yield Cre-N-Intein-N. In a similar manner, the C terminal fragment (Cre-C) is linked to the intein-C which contains 36-aa intein sequence, in the order of Intein-C-Cre-C. For testing the split-intein tagged split-Cre in cultured cortical slices, the Cre-N-Intein-N or Intein-C-Cre-C fragment was sub-cloned into pCAGGS expression vector (Addgene).
DNA sequences of the split-intein-tagged split-Cre
Generation of split-Cre transgenic mice
The Cre-N-Intein-N or Intein-C-Cre-C fragment was inserted into the unique NcoI site of the Hsp68LacZ gateway vector containing the human enhancers of interest, and a small promoter of the heat-shock protein 68 as well as the lacZ reporter gene, respectively11
. Linearized transgenes were submitted to the Duke University transgenic facility for pronuclear injection to obtain transgenic founders. All animal procedures were performed according to protocols approved by The Duke University Institutional Animal Care and Use Committee.
X-Gal staining, AP staining, and in situ hybridization
were performed following previously described protocols12,13,17
For whole-mount X-gal staining, embryos were dissected out from timed-pregnant female and fixed in 4% PFA/PBS on ice for 30 mins, rinsed with buffer A (100 mM phosphate buffer, pH 7.4, 2 mM MgCl2 and 5 mM EGTA) twice, for 5 min and 30 min each at room temperature, and followed by two washes of 5 min at room temperature with buffer B (100 mM phosphate buffer, pH 7.4, 2 mM MgCl2, 0.01% sodium desoxycholate, and 0.02% Nonidet P40). Embryos were stained for blue color by exposure in the dark at 37C in buffer C (buffer B, plus 5 mM potassium-ferricyanide, 5 mM potassium-ferrocyanide, and 1 mg/ml of X-Gal). For sections, dissected embryos were post-fixed in 4% PFA/PBS on ice for 1 hour, and saturated in 30% sucrose/PBS at 4°C overnight. Sections were collected with a cryostat at 20–25 μm thickness, and staining was conducted same as described for the whole-mount staining.
For AP staining to detect PLAP activity, sections were collected using a cryostat at 20~40 μm thickness. The sections were post-fixed with 4% PFA/PBS at room temperature for 1 hour, followed by inactivation step at 65°C for 6 hours in PBS. For the detection of AP activity, the sections were rinsed with wash buffer (0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl) and then developed in staining solution [1:50 NBT/BCIP stock solution (Roche), 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl2].
For fluorescent two-color in situ hybridization, first the cDNA sequences for the CreN and CreC fragment were used as template for in vitro transcription to generate either digoxigenin-UTP (Roche) or FITC-UTP (Roche) labeled antisense probes for CreN or CreC, respectively. The two-color in situ hybridization was performed as described previously17
Multiple E15.5 embryos from CreN752; CreC1163, RosaPlap/+ were used for two-color in situ hybridization experiments, X-gal staining, or AP staining. Trigeminal ganglia were serial sectioned onto alternating slides for X-gal and AP staining, so adjacent section pairs can be compared. 15 randomly selected sections or alternating section pairs from each type of experiments were used for counting the number of CreN+, CreC+, or CreN+CreC+ (double positive), as well as LacZ+, or Plap+ neurons. The average percentage of CreN+CreC+ double positive neurons out of the total CreN+ and/or CreC+ neurons was calculated. Similarly, the average percentage of Plap+ neurons over the total number of LacZ+ neurons was calculated.