The pronounced effects of null mutations in the myostatin gene have been well documented across species, and include hypertrophy and hyperplasia of muscle fibers (McPherron and Lee, 1997
; Berry et al., 2002
; Schuelke et al., 2004
; Clop et al., 2006
; Mosher et al., 2007
; Shelton and Engvall, 2007
; Boman et al., 2009
). Cattle that possess a null mutation in the myostatin gene, such as Belgian Blue and Piedmontese, have an increase in calving difficulty and a high degree of offspring mortality (Bellinge et al., 2005
). The ability to control the degree of suppression of myostatin in livestock may alleviate some of the negative attributes of null mutations, while at the same time increasing muscle mass and therefore meat production.
The siRNA sequences tested here were successful in suppressing myostatin expression, both when transfected in vitro as an siRNA or when expressed from a plasmid as an shRNA. A similar approach has been previously reported in zebrafish and mice, where an siRNA targeting the myostatin gene was then incorporated as an shRNA into a plasmid for in vivo transgene delivery (Magee et al., 2006
; Lee et al., 2009
). Therefore, we believe this approach should be effective when transitioning from in vitro testing to transgenic livestock production. In addition, the use of RNAi can add an additional level of control, as the degree of gene suppression could be manipulated through the choice of the shRNA that is introduced.
Currently, the most common method for producing transgenic livestock involves genetic modification of cell lines followed by the utilization of these for SCNT (cloning). Although effective, animal cloning remains an inefficient process, as highlighted by our experiences with this project, which resulted in a single transgenic calf that died at birth. In addition, issues involving intellectual property rights can limit the utilization of cloning for transgenic animal production. Clearly there is a great need for alternative methods for producing transgenic livestock that are effective, efficient, and economical. Difficulties with cloning prompted our use of lentiviral microinjection for transgenic embryo production. Previous work by Hofmann et al. (2003
) reported an efficiency of 45% GFP-positive blastocysts when bovine zygotes were microinjected 18 hr post-fertilization, and an even a higher success rate (92%) with transduction of oocytes followed by in vitro fertilization. Milazzotto et al. (2010
) also reported the injection of oocytes with lentivirus carrying a gene encoding an shRNA designed to silence the expression of myostatin. After fertilization, however, only 3.07% of the hatching blastocysts were GFP-positive (Milazzotto et al., 2010
). In the current study, microinjection of lentivirus into zygotes 8–10 hr post in vitro fertilization resulted in 78% of the blastocysts expressing GFP. Additionally, transfer of transgenic blastocysts produced through microinjection produced a similar pregnancy rate as SCNT at 35 days gestation (50%), but resulted in the live birth of 7 transgenic calves versus 0 from SCNT. This clearly demonstrates the increased efficiency of transgenic animal production with this method over cloning.
The constructs used in these experiments were designed such that transcripts encoding the GFP reporter and the shRNA were produced as a single messenger RNA (). Accordingly, GFP transcription and fluorescence levels are both strong indicators of shRNA production (Golding and Mann, 2011
). In these studies, analysis of transgene expression in embryos, fetuses, and live calves involved both observations for GFP fluorescence and/or quantitative real-time PCR analysis of GFP mRNA. Blastocysts were confirmed to be expressing the transgene prior to embryo transfer. Collected fetuses also expressed the transgene, as did the cloned calf and at least four of the live calves produced by injection of lentivirus into zygotes (3 derived using MSTN-1026 shRNA and 1 NULL control).
Figure 4 Graphic representation of the lentiviral plasmids used in this experiment. A: Lentiviral plasmid used to produce transgenic donor cells for somatic cell nuclear transfer. This plasmid contains a puromycin resistance gene for antibiotic selection of transgenic (more ...)
Considerable variation in transgene expression levels was observed between different calves, and expression of the transgene was undetectable in at least two cases. The significance of this, as related to our overall goals for this project, is tempered by the likelihood that transgene expression varies between animals relative to their developmental clock. Previous studies by our group and others have suggested the EF1α promoter used in these constructs is both highly expressed and epigenetically stable in embryonic and extraembryonic tissues (Golding and Mann, 2011
). Furthermore, transgenic mice produced through sub-zonal injection of lentiviral particles exhibited strong transgene expression in a variety of tissues over six generations (Kvell et al., 2010
). The strength and stability of this promoter in cattle, however, has not been examined until now. Myostatin mRNA is normally produced by the developing somites and is detected by RT-PCR as early as day 29 in cattle (Kambadur et al., 1997
). Primary myotube development is established by day 39 of gestation in cattle, and secondary myofiber differentiation is established by day 90 (Oldham et al., 2001
). Therefore, it is expected that expression of transgenes designed to silence the expression of myostatin during this particular time of gestation would result in increased muscle development in the resulting fetuses and calves. We confirmed that embryos were expressing the transgene at the time of embryo transfer, but we do not know the status of gene expression during fetal development of the transgenic calves. The possibility exists that those calves that did not exhibit gene expression after birth did express the transgene during fetal development.
depicts two transgenic bulls, one with the gene encoding shRNA MSTN-1026 (R181) and the second encoding the NULL shRNA (R170, control). Both of these bulls are 14 months old and both were derived from the same Brangus sire. The transgenic bull encoding MSTN-1026 obviously appears to exhibit more muscle mass when compared to the transgenic NULL. This bull also exhibits the lowest level of myostatin mRNA when compared to other bulls; unfortunately, we were unable to detect transgene expression in muscle derived from this animal (B). The control bull (R170) expressed the transgene encoding a nonsense shRNA. The increased muscle development exhibited in bull R181 might represent an example where the shRNA targeting myostatin was expressed during fetal development and not in the adult. At this point, however, there is no way to resolve this uncertainty. To date we have not produced enough transgenic calves to demonstrate any association between transgene expression and phenotype. Analysis of muscle samples derived from the transgenic calves does not support a correlation between expression of MSTN-1026 and myostatin, even though in vitro studies clearly demonstrate this shRNA sequence to be highly effective at silencing myostatin gene and protein expression.
Figure 5 Genetically modified calves with a transgene encoding (A) shRNA MSTN-1026 targeting the myostatin gene or (B) a NULL shRNA at 14 months of age. The same Brangus sire was used for in vitro fertilization in the production of these two calves; the genetic (more ...)
All the calves produced in these studies were derived from ova collected from a slaughterhouse, and we have no information on their dams beyond the general genetic background of cows normally processed at this facility being of dairy breed descent (Holstein). Differences in muscle development exhibited between these two animals could be explained by their genetic background (parents) or by environmental/epigenetic factors. Additional experiments will have to be conducted and more animals produced to thoroughly test this hypothesis.
In conclusion, here we report the production of transgenic calves expressing an shRNA that effectively reduces myostatin mRNA and protein expression in vitro. Both SCNT and lentiviral microinjection resulted in a high percentage of embryos expressing the transgene, although lentiviral microinjection proved more efficient in the production of live offspring. Work is currently underway to use these cattle for breeding studies to determine the inheritance patterns of the transgene, in addition to gene expression and the extent of myostatin suppression and muscle characteristics in these animals. Production of transgenic livestock expressing an shRNA targeting the myostatin gene should result in animals with enhanced muscle development. This could increase the efficiency of food production agriculture, and analysis of such animals could also contribute to the development of human therapeutics for the treatment of muscle wasting disorders.