| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1,2
* Department of Biotechnology, Institute for Animal Breeding (FAL), Mariensee, Germany;
Klinik für Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany; and
Molecular Biotechnology, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany
2Correspondence: K. W., Klinik f. Viszeral-und Transplantations Clinique, Medizinische Hochsule Hannover, Carl-Neuberg Str. 1, Hannover D-30623, Germany. E-mail: wonigeit.kurt{at}mh-hannover.de H. N., Institut for Animal Breeding (FAL) Department of Biotechnology Höltystr. 10 Neustadt D-31535, Germany. E-mail: niemann{at}tzv.fal.de
SPECIFIC AIMS
The original tetracycline system requires two DNA constructs for constitutive expression of the transactivator and the transactivator-dependent expression of the target gene, respectively, and has been successfully used in mice where both elements are integrated in two different transgenic lines. On breeding, offspring are obtained in which expression of the target gene can be regulated by addition of doxycycline. The long generation intervals make this approach unfeasible in other mammalian species, including livestock. One approach to overcome this limitation is to combine both transactivator and target genes, including individual promoters in a single construct. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. The specific aims of this study were: 1) to demonstrate that single-construct tet-cassettes can be successfully expressed in animals, and 2) and to improve xenotransplantation by producing tet-off regulated human complement regulator (hCD59, hDAF) transgenic animals. The following principal findings were made:
1. Functionality of autoregulatory RCA–NTA expression cassettes in cell lines
The autoregulatory bicistronic tet-off based cassettes (Fig. 1
A) driven by the tet-responsive promoter (PtTA) showed autoactivation and expression of the respective RCA (Fig. 1B
) in mouse NIH 3T3 cells and swine testis epitheloid (STE) cells. Expression was efficiently suppressed by addition of doxycycline to the culture medium (Fig. 1B
). Thus, autoregulatory bicistronic expression cassettes support tight regulation of human RCAs in xenogenic cells.
|
2. Characteristic expression pattern in RCA-NTA transgenic pigs
In the absence of any tetracycline, Northern blotting and immunohistology revealed the presence of mRNA and protein for decay accelerating factor (DAF) or CD59 in single fibers of striated muscle in nine of the 10 transgenic lines (Fig. 1C
) derived from microinjection. Depending on the line, 5–30% of the muscle fibers stained positive. The animals did not express human RCAs in brain, heart, kidney, tongue, liver, skin, lymph nodes, blood, and pancreas. Apparently, the (different) integration sites of the nine expressing lines had no or only a marginal effect on the muscle fiber-specific expression. The entire bicistronic cassettes were integrated into the porcine genome as shown by Southern blotting of genomic DNA and polymerase chain reaction. Fodder and plasma samples of transgenic pigs were negative for tetracycline activity, ruling out potential contaminations.
3. Enhanced tissue-independent expression of CD59 in crossbred animals carrying two NTA-RCA cassettes
A selective up-regulation of CD59 expression was found in double-transgenic animals derived from crossbreeding animals carrying either CD59-NTA or DAF-NTA. The CD59 mRNA levels were significantly (50–100 fold) elevated in three different muscles, i.e., Musculus (M.) longissimus, front and hind leg muscles, in double-transgenic animals over transgenic siblings carrying only one CD59-NTA cassette. Virtually all fibers in striated muscle stained positive for CD59 protein. Other organs, including liver, pancreas, kidney, and lung expressed the transgene at the mRNA and protein levels. RNA and protein expression pattern of CD59 correlated well. In all examined tissues, the positive cells were strongly stained for CD59, suggesting that the autoregulative up-regulation worked well in this population of cells. However, DAF mRNA and protein levels were not increased in the same double-transgenic animals and immunoreactivity remained confined to single muscle fibers. Seven out of nine double-transgenic pigs, derived from crossing two transgenic lines, showed this asymmetric CD59 over-expression. In the remaining two double-transgenic lines of pigs, expression patterns mimicked that of their parental lines showing mosaic expression in single muscle fibers and individual muscle fibers coexpressed both CD59 and DAF, while some fibers expressed only CD59, but not DAF. Fibers with exclusive expression of DAF were never detected. Since none of the single-transgenic animals showed this enhanced transgene expression, it is suggested that the presence of two cassettes is necessary, but not always sufficient, for tissue-independent expression. Health status, development, and fertility of animals bearing either one or two NTA-cassettes were not compromised. All animals were not distinguishable from their nontransgenic counterparts.
4. Conditional RCA-NTA regulation in single- and double-transgenic animals
Single-transgenic and double-transgenic animals were fed with doxycycline for 6 d and muscle biopsies taken before, during and after antibiotic treatment showed an effective shut down of transgene expression. RCA-NTA transcripts were virtually eliminated by doxycycline feeding within 2–6 d. Thus, the expression cassettes located at different chromosomal sites could be readily switched off. Re-expression of RCA-NTAs did not resume until eight weeks after termination of the doxycycline application.
5. Tightly controlled expression of human RCA in white blood cells
Flow cytometry revealed no significant expression of DAF or CD59 in resting and concanavalin A-activated lymphocytes from single-transgenic pigs. Absence of DAF and CD59 was also typical for resting cells from double-transgenic animals. However, expression of DAF and CD59 could be induced in cells from double-transgenic animals by activation with ConA. The intensity of ConA-mediated up-regulation of the two molecules was asymmetric showing strong expression of CD59 and only weak up-regulation of DAF. Expression of both molecules could be terminated by culturing the cells with tetracycline for 48 h, providing additional evidence for a tight control of gene expression from these cassettes.
6. Identification of CpG islands and determination of the methylation status
Bioinformatic analysis of the DAF-NTA cassette revealed that the minimal cytomeglovirus (CMV) promoter within PtTA and the 5'coding region of the DAF cDNA together contained a putative CpG island, while no CpG island was predicted for the PtTA and the 5'coding region of the CD59 gene. To unravel potential epigenetic mechanisms involved in silencing of the transgene, bisulfite sequencing using genomic DNA from muscle biopsies was used and showed that the promoter region of the CD59-NTA cassette was completely methylated in single-transgenic animals, while in double-transgenic animals only 
50% of the CpG dinucleotides were methylated. The CpG dinucleotides around the transcription initiation site of the DAF-NTA cassette were also methylated in single- and double-transgenic animals. Stably transfected NIH 3T3 cells and two cell clones, which expressed high levels of the DAF-NTA construct possessed a low concentration of methylation. Stably transfected porcine STE cells showed weak methylation of the promoter regions. This indicates an inverse correlation between the observed gene expression and the methylation status of these cassettes. The PtTA is prone to methylation in transgenic pigs, in particular in combination with the 5'coding region of the human DAF gene.
CONCLUSIONS AND SIGNIFICANCE
Here, we have generated transgenic pigs bearing a bicistronic tetracycline-responsive cassette, and demonstrated tightly controlled expression in a large animal model for the first time (Fig. 2
). The usage of the autoregulative bicistronic cassette supports efficient single step transduction, which is of utmost importance in view of the long generation intervals in large domestic animals. Expression of RCAs and transactivator did not compromise health status and fertility of transgenic pigs. Design of the next generation of expression cassettes will take into account potential methylation prone sequences and should thus be compatible with true ubiquitous transgene expression already in the F0-generation. The use of optimized autoregulative constructs in combination with improved gene transfer techniques, such as somatic nuclear transfer or lentiviral vectors will pave the way toward precisely controlled transgene expression in farm animals for biomedical and agricultural application.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5415fje
1 Present address: Envelope retrovirale et ingeniérerie retrovirale, INSERM 758, ENS de Lyon, France. 
This article has been cited by other articles:
|
|
 |
|
  F. Park Lentiviral vectors: are they the future of animal transgenesis? Physiol Genomics, October 19, 2007; 31(2): 159 - 173. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
