Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. We have assessed the α-casein (CSN1S1) gene as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression. This article is protected by copyright. All rights reserved.</p

Arginine to glutamine variant in olfactomedin-like 3 (OLFML3) is a candidate for severe goniodysgenesis and glaucoma in the Border Collie dog breed

Goniodysgenesis is a developmental abnormality of the anterior chamber of the eye. It is generally considered to be congenital in dogs (), and has been associated with glaucoma and blindness. Goniodysgenesis and early-onset glaucoma initially emerged in Border Collies in Australia in the late 1990s and have subsequently been found in this breed in Europe and the USA. The objective of the present study was to determine the genetic basis of goniodysgenesis in Border Collies. Clinical diagnosis was based on results of examinations by veterinary ophthalmologists of affected and unaffected dogs from eleven different countries. Genotyping using the Illumina high density canine single nucleotide variant genotyping chip was used to identify a candidate genetic region. There was a highly significant peak of association over chromosome 17, with a -value of 2 × 10 Expression profiles and evolutionary conservation of candidate genes were assessed using public databases. Whole genome sequences of three dogs with glaucoma, three severely affected by goniodysgenesis and three unaffected dogs identified a missense variant in the olfactomedin like 3 () gene in all six affected animals. This was homozygous for the risk allele in all nine cases with glaucoma and 12 of 14 other severely affected animals. Of 67 reportedly unaffected animals, only one was homozygous for this variant (offspring of parents both with goniodysgenesis who were also homozygous for the variant). Analysis of pedigree information was consistent with an autosomal recessive mode of inheritance for severe goniodysgenesis (potentially leading to glaucoma) in this breed. The identification of a candidate genetic region and putative causative variant will aid breeders to reduce the frequency of goniodysgenesis and the risk of glaucoma in the Border Collie population

Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

AbstractTraditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals

Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions

Analysis of milk calcium and phosphate levels and milk protein gene expression.

<p><b>Panel A:</b> Calcium and phosphate content of mouse milk was determined as indicated in the methods section. Concentrations are given in nM. <b>Panel B:</b> Quantitative PCR analysis of α-casein and β-casein gene expression. cDNA derived from representative wild-type, heterozygous [+/−] and homozygous [−/−] α-casein deficient mice was analysed using primer pairs specific for α-casein, β-casein and the reference gene GAPDH. Expression of the casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel C:</b> Quantitative PCR analysis of γ-casein and κ-casein gene expression. Expression of the γ and κ-casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel D:</b> Correlation of casein gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Casein gene expression was correlated with the expression of the reference gene β-actin. Quantification of α-casein was done in 3 [+/+], 7 [+/−] and 5 [−/−] mice. Quantification of β-casein was done in 3 [+/+], 8 [+/−] and 4 [−/−] mice. Quantification of γ- and k-casein was done in 3 [+/+], 3 [+/−] and 3 [−/−] mice. Expression in heterozygous and α-casein deficient mice is presented as percentage of median casein gene expression in wild-type control mice [+/+] (set to 100%). Error bars represent standard deviations. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, p<0.01 by **, and p<0.001 by ***. Exact p values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021775#pone-0021775-t006" target="_blank">table 6</a>.</p

Comparison regarding general health and behaviour between pups nursed by wild-type dams and pups nursed by α-casein deficient dams.

<p>P-values of parameters without significant differences between group 1 (G1: wild-type pups nursed by wild-type dams) and 2 (G2: wild-type pups nursed by α-casein deficient dams; P-value G1 vs. G2) and group 1 and 3 (G3: heterozygous pups nursed by wild-type dams; P-value G1 vs. G3) at 8 weeks of age (Fisher's exact test and *Mann-Whitney-U test). Constant values indicate that all animals in the groups compared had the same, normal, score.</p

Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.

<p><b>Panel A:</b> Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) <b>Panel B:</b> Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. <b>Panel C:</b> Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.</p