Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM+ and IgG+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.
A leucine/valine substitution of growth hormone amino-acid 127 was genotyped in 221 Bavarian Simmental bulls by PCR/RFLP. Gene frequencies for leucine (L) and valine (V) were 0.68 and 0.32, respectively. Breeding values for meat traits were significantly different between genotypes. The heterozygous genotype LV was superior to LL and VV in both carcass gain (p < 0.01) and meat value, an index which includes carcass gain and classification score (p < 0.05). In relation to classification score, genotype VV was significantly better (p < 0.05) than LV and VV. No effects on milk breeding values were apparent even though those of milk composition approached significance. ZUSAMMENFASSUNG: Einfluß von Wacbstumshormon-Genotypen auf Zuchtwerte von Fleckviehbullen Ein Polymorphismus der Aminosäure 127 des bovinen Wachstumshormones wurde bei 221 bayerischen Fleckviehbullen mit PCR/RFLP genotypisiert. Die Genfrequenzen für Leucin (L) und Valin (V) betrugen 0.68 und 0.32. Es bestand ein signifikanter Einfluß auf die Fleischzuchtwerte. Der heterozygote Genotyp LV war in den Merkmalen Nettozunahme (p < 0.01) und Fleischwert (p < 0.05) den Geno-typen LL und VV signifikant überlegen. Im Merkmal Handelsklasse war Genotyp VV signifikant günstiger als LV und LL. Es konnten keine Beziehungen zu den Milchzuchtwerten beschrieben werden, allerdings grenzte der Einfluß auf Milchzusammensetzung an statistische Signifikanz.
A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.
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