Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice

Abstract

Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin kappa gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum kappa chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.

Fig. 1

Map of plasmid DNA pB1–14. The functional MOPC-21 κ gene was initially cloned from genomic DNA into Charon 4A (M21B1). The V_κM.21–C_κ gene insert was excised at the EcoRI sites and cloned into the EcoRI site of pBR322. Because injection of linear DNA with heterologous ends seems to increase the likelihood of integration (R. L. B. and R. Palmiter, unpublished), such a fragment was restricted from plasmid pB1–14 using BglI and XhoI. The large BglI*–XhoI* fragment (14.8 kb) was separated from the smaller BglI–BglI and BglI–XhoI fragments by sucrose gradient centrifugation, ethanol precipitated, redissolved in 10 mM Tris pH 7.5, 0.25 mM EDTA and injected. V_L, leader sequence; VMOPC-21, variable region; J2 to J5, joining segments; C_κ, constant region. Probes for V_κM.21 (pES205) and C_κ (M13–C_κ are indicated. EcoRI**, the unique EcoRI site present in the microinjected pB1–14 gene. The 5.3 kb, 2.0 kb and 2.9 kb BamHI restriction fragments obtained by hybridization with pBR322 and C_κ (see Fig. 2b) are indicated; the broken line denotes the portion (5.9 kb) which was eliminated from the plasmid DNA before microinjection. The fact that a 2.9-kb long pBR322 positive BamHI restriction fragment was obtained in DNA of transgenic mice (Fig. 2b) suggests that the free ends of the linear plasmid DNA are joined in the integrated, amplified pB1–14 DNA.

Fig. 2

DNA analysis of transgenic mice (same mice as in RNA and protein analyses in the following figures), a, Quantitation of the microinjected gene. The dot hybridizations were probed with pBR322. Each row (top to bottom) contains 0.2, 1.0, 5.0 µg DNA. 1–3, Normal mouse DNA with 1.5, 7.3 or 29.3 copies, respectively, of 1,000 base pairs (bp) of pBR322 added per cell; 4–8, kidney DNA from transgenic mice: offspring of father A (4, 5), B (6), C (7, 8); 9–11, kidney DNA from normal littermates A, B, C, respectively, b, Southern blot. Each lane contains 30 µg of kidney DNA digested with BamHI. The blot was probed with C_κ and pBR322. 1, 2, Positive mice of transgenic line A; 3, line B; 4, 5, line C. E₁ and E₂, endogenous κ genes of C57BL/6 and 5JL mice respectively. →, Bands expected from BamHI digestion of plasmid pB1–14 when tandemly integrated in head to tail fashion (see Fig. 1). When the same blot was probed with pBR322 alone, only the 2.9 and 2.0 kb bands were seen (not shown), c, Southern blot. Each lane contains 30 µg of kidney DNA (except lane 5 which contains 30 µg of tail DNA), digested with EcoRI. The blot was probed with pBR322. 1–5, As in b.

Fig. 3

RNA blots of transgenic mice and normal littermates. Poly(A)⁺ RNA was prepared from spleens (S) and livers (L) and 0.5 µg (a) or 1 µg (b, c, d) after denaturation with gly-oxal were spotted onto nitrocellulose. The spots were hybridized with the following probes: M13 C_κ (contains about 500 bp of C_κ and 3′ untranslocated region subcloned into M13) (a); pES205 (contains 103 bp of the V_κ region of MOPC-21 subcloned into pBR322) (b); pBR-322 (c); cDNA prepared from total liver poly(A)⁺ RNA (d). Hybridization conditions were 0.45 M NaCl, 0.045 M Na citrate 5× Denhardt’s solution, 0.1% SDS, 50 µg ml⁻¹ yeast tRNA (Sigma), 2.5 µg ml⁻¹ poly(A), 50 µg ml⁻¹ heat-denatured salmon testis DNA, 25 ng ml^{−1 32}P-labelled, nick translated, heat-denatured probe, 10 ml total volume per nitrocellulose filter, 67 °C, 24 h. The specific activity of the probes was about 2× 10⁸ c.p.m. µg⁻¹. After hybridization the filters were washed exhaustively in 15 mM NaCl, 1.5 mM Na citrate, 0.05% SDS at 65 °C. A, B, C represent three different transgenic mouse lines. Each subscript number represents an individual mouse. +, Transgenic mice, −, negative littermates. X, spot of probe DNA. Exposures to Kodak X-omat film were for 4 days (a), 2 days (b), 5 days (c), 12 h (d).

Fig. 4

Northern blot of total glyoxal treated spleen RNAs (30 µg per lane) of a pool of five normal mice (1, 5 and 9) and pools of three, five and three positive offspring from three different transgenic mice respectively (2, 6 and 10; 3, 7, and 11; 4, 8 and 12). Hybridization conditions were as in Fig. 3; probes were M13 C_κ (1–4) and pES205 (V_κM.21) (5–12). Sizes of 28S and 18S ribosomal RNAs were determined from total mouse myeloma RNA electrophoresed in the same gel and then stained with ethidium bromide. After hybridization with C_κ the blot of 1–4 was treated in 90% formamide at 65 °C for 3 h to remove most of the probe and later rehybridized with V_κM.21 (tracks 5–8). The small amount of radioactivity in lane 5 is from some remaining C_κ probe which gave the same signal after reexposure to X-ray film before hybridization with V_κM.21 (not shown). The RNAs of tracks 9–12 were electrophoresed on a different gel and hybridized only with V_κM.21.

Fig. 5

Relative quantities of C_κ and V_κM.21 RNAs in spleens of transgenic mice and normal littermates. a, Probed with M13 C_κ. The spleen RNAs were 200 ng, 400 ng and 800 ng. b, Probed with pES205. The spleen RNAs were 0.5 µg, 1 µg and 2 µg. Methods: Spleen poly(A)⁺ RNAs of the two positive offspring A were pooled, as were the poly(A)⁺ RNAs of offspring C. As controls, spleen RNA of negative A and C offspring were pooled together. Dot hybridizations of these RNAs included as a standard the DNA of the M21B₁ clone, and were hybridized with a C_κ or V_κM.21 probe. A⁺: pool of spleen poly(A)⁺ RNAs of transgenic mice 1 and 2 (see Fig. 3); C⁺: pooled spleen poly(A)⁺ RNAs of transgenic mice 6 and 9. AC⁻: pooled spleen poly(A)⁺ RNAs of negative litttermates 3, 7 and 8. PH, DNA of phage clone M21B1 (see Fig. 1), 1, 2 and 4 ng (in a), 10, 20 and 40 ng (in b). Exposure to X-ray film: 14 h.

Fig. 6

Two-dimensional (2-D) gels of serum proteins, a, Purified MOPC-21 immunoglobulin (Litton Bionetics). b and c, 25 µl of serum of + and − offspring A, respectively, d, Additional examples of the characteristic 2-D gel regions of serum from one transgenic mouse (+) and five normal littermates (−). The sera were precipitated with 40% ammonium sulphate, the precipitate redissolved in glass distilled water and run on 2-D gels after O’Farrel gels which were silver stained. Molecular weights (MW) of marker proteins run in the second dimension of the same gel (a) or of a control gel (b, c) are indicated.