Genetically modified rabbits as bio-producers and biomodels

Genetically modified (GM) animals are necessary to solve the global problems of humanity related to nutrition and health. Rabbits, as laboratory, domestic and farm animals, occupy a special niche in research. GM rabbits are promising as bioreactors for producing biologically active (BA) proteins with milk or blood, and are in demand in Biomedicine as biomodels of diseases. To date, many GM rabbits-biomodels, producers of recombinant proteins have been created in the world using CRISPR/Cas9 technology. All-Russian Research Institute of Animal Physiology, Biochemistry and Nutrition has experience in obtaining transgenic rabbitsproducers of human BA proteins with milk by microinjecting recombinant DNA into zygote pronuclei. The possibility of site-specific modification of the rabbit whey acidic protein (WAP) gene using CRISPR/Cas9 technology is discussed. A DNA matrix containing homology arms to the WAP rabbit gene and site-specific CRISPR/Cas9 components in plasmid form were obtained. Microinjections of rabbit zygotes were performed and embryo survival was evaluated in vitro. The efficiency of using the green fluorescent protein gene under the cytomegalovirus promoter in the DNA matrix as an indicator of homologically directed repair was evaluated. This work can be useful for obtaining rabbits that produce with milk BA protein instead of WAP.

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p<0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not siginificantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

Cloning of the gene for interstitial collagenase-3 (matrix metalloproteinase-13) from rabbit synovial fibroblasts: differential expression with collagenase-1 (matrix metalloproteinase-1)

Cartilage, bone and the interstitial stroma, composed largely of the interstitial collagens, types I, II and III, are remodelled by three members of the metalloproteinase (MMP) family, collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13). MMP-1 and MMP-13 may contribute directly to disease progression, since they are induced in patients with rheumatoid arthritis and osteoarthritis. The study of MMP-1 and MMP-13 gene regulation in models of arthritic disease has been problematic because mice and rats, which are typically used, only possess a homologue of MMP-13. Here we show that in contrast with mice and rats, rabbits possess distinct genes homologous to human MMP-1 and MMP-13. Furthermore, rabbit MMP-13 is expressed simultaneously with MMP-1 in chondrocytes and synovial fibroblasts in response to the cytokines interleukin-1 and tumour necrosis factor-α, or the phorbol ester PMA. The time course of MMP-13 induction is more rapid and transient than that of MMP-1, suggesting that distinct mechanisms regulate the expression of these two collagenases. We have cloned the rabbit MMP-13 gene from synovial fibroblasts and demonstrated that the rabbit gene shares greater homology with human MMP-13 than does the mouse interstitial collagenase. Together with the fact that mice and rats do not possess a homologue to human MMP-1, our data suggest that the rabbit provides an appropriate model for studying the roles of interstitial collagenases in connective-tissue diseases, such as rheumatoid arthritis and osteoarthritis.

Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis)

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.