Article Figures & Data
Figures
- FIGURE 1.
Structure and copy number analysis of transgenes. A, Schematic diagram of the murine heavy chain locus and the DNA fragments that were cloned for transgenic preparation. Regulatory elements are depicted as ovals, exons are dark hatched rectangles, and switch regions as light hatched rectangles. B, BamHI; S1, SalI; SII, SstII; E, EcoRI; P, PstI. B, Representative Southern blot used in the verification of transgene insertion and the determination of transgene copy number. BamHI-digested tail DNA was analyzed using the BamHI/SstII fragment from the 3′ end of the ISCγ2a cloned fragment. Head-to-tail insertions of the ISCγ2a fragment are revealed by hybridization as a 5.2-kb BamHI fragment (4.7 kb from the tail + 0.5 kb from the head). Alternating, head-to-tail insertions of the ISCγ2a + either HS1234 or Eμ are revealed as a 4.7-kb BamHI fragment (the BamHI site is in the polylinker at the head of the HS1234 or Eμ fragments). Copy number was determined by quantitation on a PhosphorImager, with the endogenous band (“Endg.”) as a standard, 2 gene copies.
- FIGURE 2.
The ISCγ2a transgene is insertion site independent and copy number dependent. RT-PCR was performed with RNA extracted from T-depleted splenocytes harvested from various transgenic mice (copy numbers as indicated) after 3-day cultures in the presence of LPS, with or without IFN-γ. “Relative expression” is the ratio of the amount of transgenic PCR product to the amount of endogenous PCR product, as determined by densitometry of the x-ray film. The asterisked values were too small to accurately determine. The relative expression of 0.60 in line 4606 is anomolously high due to false signal on the left-hand side of the lane.
- FIGURE 3.
The ISCγ2a transgene is expressed in a cytokine-inducible and cell-type specific manner. A, RNA from 3-day cultures of T cell-depleted splenocytes or fresh thymocytes was analyzed by RT-PCR. B, Semiquantitative radiolabeled PCR. Three (4601 samples) or four (131 samples) 5-fold dilutions of cDNA were amplified for the indicated mRNAs from T-depleted splenocytes treated as indicated. The relative expression values, as defined in the legend for Fig. 2, for the 4601 LPS + IFNγ samples were 0.020–0.026.
- FIGURE 4.
Expression of ISCγ2a transgenes is increased 25- to 75-fold when linked to enhancers from the murine heavy chain locus. Iγ2a and actin transcripts were detected by an S1 nuclease protection assay. A, RNA was prepared from total splenocytes (RBC-lysed) cultured for 3 days with the indicated additions. The RNA analyzed in lane 11, however, was prepared from fresh (not cultured) splenocytes. Transgenic donors of the splenocytes all carried the ISCγ2a transgene linked to Eμ or HS1234 (as indicated) and are identified by founder number (275, etc.). Transgene copy numbers are indicated. B, RNA was prepared from fresh total splenocytes, fresh thymocytes, or RBC-lysed splenocytes which had been cultured for 3 days with LPS or LPS + IFN-γ. All cell samples are from line 4443 (ISCγ2a + HS1234). C, Transcription start site analysis with S1 nuclease. A total of 40 μg nontransgenic or 10 μg transgenic RNA was hybridized to JCA233 for S1 nuclease analysis. The 5′-most start site in the nontransgenic sample (lane 1) was defined as “+1” and other start sites are indicated by nucleotide distance relative to it (left side of the panel). The probe extends 69 bp 5′ and 164 bp 3′ of +1, and the approximate migration position of full length protection of the 233-bp insert is indicated (“FL”). +1 and +51 correspond to the two major transcription start sites in 18.81A20 cells (25 ). The material at the top of each lane is undigested probe, or probe that has been “nibbled” into the M13 polylinker portion.
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