Assessment of fecundity and germ line transmission in two transgenic pig lines produced by sleeping beauty transposition

Abstract

Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome.

Figure 1

Potential combinations of Sleeping Beauty components. (A) Cytoplasmic injection of an expression plasmid encoding Sleeping Beauty transposase and a plasmid carrying a SB-transposon; (B) Cytoplasmic injection of Sleeping Beauty-mRNA and a SB-transposon; (C) Injection of recombinant Sleeping Beauty protein and a SB-transposon; (D) In all possibilities stable transposition into a chromosome occurs, however, it is likely that A-C differ in the kinetics and potential remobilization events. CMV-SB100x, cytomegalovirus immediate early promoter driving a SB100x-cDNA; CAGGS, cytomegalovirus enhancer, chicken beta actin hybrid promoter driving a Venus-cDNA; ITR, SB specific-inverted terminal repeat; blue triangles, heterospecific loxP sites for a potential recombinase-mediated cassette exchange [25].

Figure 2

Transposon transgenic pig expressing Venus reporter. (A) Boar #505 under specific Venus excitation at the age of four weeks, Venus fluorescence is indicated by green color. Note the blue appearance of the hands holding the pig is due to reflected and scattered excitation light; (B) Scheme of CPI injection. After injection of the plasmid solution into the cytoplasm (I.), the plasmids translocate into the nucleus (II.). Expression of the SB100× helper plasmid results in the production of active SB protein and specific transposition of the transposon (without plasmid backbone) into the genome (III.).

Figure 3

Expression of Venus fluorescence in spermatozoa. (A) Specific Venus fluorescence of a spermatozoon from F0 boar #503; (B) Brightfield image of A; (C) Flow cytometric analysis of pig spermatozoa. The black line in the FACS analysis represents wild type sperm; blue line, spermatozoa from F1 boar with single integration; red line, spermatozoa from boar #503 with three integrations. Note that all spermatozoa from the transgenic pigs are uniformly Venus-positive.

Figure 4

Expression of Venus fluorophore in the pig heart and absence of Venus in erythrocytes. (A) Exemplarily, a transgenic (left) and a wild type (right) pig heart are shown under specific excitation of the Venus fluorophore. Inset, brightfield view; (B) Smear of blood cells. Brightfield view (left), specific Venus excitation (right). Note, red blood cells showed no Venus expression (some erythrocytes are indicated by an arrow); (C) Western blot detection of Venus in blood fractions. In blood, the Venus expression is restricted to leucocytes. Green bar indicates samples from Venus transgenic pig; black bar samples from wild type pig. M, molecular size marker; cb, complete blood; p, plasma; e, erythrocyte fraction; l, leucocyte fraction. Apparent molecular weight of Venus protein is ~30 kD.

Figure 5

Organ and cell type-specific expression of Venus. Cryosections (10 µm) of A. heart, B. liver, C. lung, D. kidney, and E. pancreas of a Venus transgenic F1 pig are shown under normalized fluorescence excitation (left) and brightfield conditions (right). Almost all cells express the Venus reporter, however at cell type-specific levels.

Figure 6

Southern blot analysis of F1 offspring. (A) Southern blot of eight F1 offspring (litter 5, Tab 1). 1×, 2× and 0× indicate the segregated transposon copy numbers. Piglets #1, #2, #3, #8 (lanes 1, 2, 3, 8) carry one copy of a monomeric transposon, piglets #5 and #7 carry two transposon copies, and #4 and #6 are non-transgenic. Red arrow: internal fragment ~1.4 kb. Green arrows: external fragments of the first and second integrant. Note: In this litter was no piglet with three transposon copies. M, marker; 1-12, lane number; - no sample was loaded; wt, wild type DNA; c, positive control; (B) Design of Southern blot. Genomic DNA was digested with NcoI, which releases an internal fragment of 1.4 kb and an external fragment of >1.4 kb depending on neighboring sequences of the transposon. The black bar indicates position of Digoxigenine labeled probe, which hybridizes to internal and external fragments. Thus each integrated transposon should result in a 1.4 and a > 1.4 kg fragment. Illegitimate integration can be excluded by employing restrictions flanking the ITR on original transposon plasmid, for details see [25].

Figure 7

Maximized Venus expression by crossbreeding. (A) Female F1 sow # 518 (descendant of boar # 503) was inseminated with semen of the F0 boar #505. Seven of the F2 offspring (Table 1, litter 6) are shown under specific Venus excitation. The piglets are labeled with the copy number of the transposon cassette. The number of integration sites of the Venus transposon shows a direct correlation to fluorescence intensity; (B) Calculated transposon segregation for parents with three and two independent integrants of the Venus transposon. Dot, Venus transposon integrant; line, wt allele.