Production of light-coloured, low heat-absorbing Holstein Friesian cattle by precise embryo-mediated genome editing.

Context: Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns.

Nanoformulated Recombinant Human Myelin Basic Protein and Rituximab Modulate Neuronal Perturbations in Experimental Autoimmune Encephalomyelitis in Mice.

Introduction: Rituximab (RTX) and recombinant human myelin basic protein (rhMBP) were proven to be effective in ameliorating the symptoms of multiple sclerosis (MS). In this study, a nanoformulation containing rhMBP with RTX on its surface (Nano-rhMBP-RTX) was prepared and investigated in comparison with other treatment groups to determine its potential neuro-protective effects on C57BL/6 mice after inducing experimental autoimmune encephalomyelitis (EAE).

Methods: EAE was induced in the corresponding mice by injecting 100 μL of an emulsion containing complete Freund's adjuvant (CFA) and myelin oligodendrocyte glycoprotein (MOG).

Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos.

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices.

Holstein Friesian dairy cattle edited for diluted coat color as a potential adaptation to climate change.

Background: High-producing Holstein Friesian dairy cattle have a characteristic black and white coat, often with large proportions of black. Compared to a light coat color, black absorbs more solar radiation which is a contributing factor to heat stress in cattle. To better adapt dairy cattle to rapidly warming climates, we aimed to lighten their coat color by genome editing.

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis.

Background: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e.

Transgenic goats producing an improved version of cetuximab in milk.

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs.

Cetuximab produced from a goat mammary gland expression system is equally efficacious as innovator cetuximab in animal cancer models.

There is increasing demand for improved production and purification systems for biosimilar or biobetter humanised monoclonal antibodies and animal production systems offer one such possibile option. Cetuximab, also known as 'Erbitux', is a humanised monoclonal antibody widely used in cancer therapy. We have previously reported on a genetically engineered goat system to produce cetuximab (gCetuximab) in milk.

Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos.

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome.

Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin.

We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth.

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome.

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration.

Taillessness in a Cloned Cow is Not Genetically Transmitted.

Somatic cell nuclear transfer (SCNT), commonly referred to as cloning, results in the generation of offspring that, except for mitochondrial DNA, are genetically identical to the nuclear donor. We previously used a genetically modified bovine cell line as the donor for SCNT and obtained a calf, named Daisy, that was born without a tail. To determine whether the missing tail was a result of the genetic modification, we performed recloning experiments by using either cells from a sacrificed pregnancy of a second clone (Daisy's "twin" clone) or cells from tailless Daisy as donors for SCNT.

KDM4B-mediated reduction of H3K9me3 and H3K36me3 levels improves somatic cell reprogramming into pluripotency.

Correct reprogramming of epigenetic marks is essential for somatic cells to regain pluripotency. Repressive histone (H) lysine (K) methylation marks are known to be stable and difficult to reprogram. In this study, we generated transgenic mice and mouse embryonic fibroblasts (MEFs) for the inducible expression of KDM4B, a demethylase that removes H3 K9 and H3K36 trimethylation (me3) marks (H3K9/36me3).

Development and Pre-Clinical Evaluation of Recombinant Human Myelin Basic Protein Nano Therapeutic Vaccine in Experimental Autoimmune Encephalomyelitis Mice Animal Model.

Unlabelled: Recombinant human myelin basic protein (rhMBP) was previously produced in the milk of transgenic cows. Differences in molecular recognition of either hMBP or rhMBP by surface-immobilized anti-hMBP antibodies were demonstrated. This indicated differences in immunological response between rhMBP and hMBP.

Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects.

We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells.

Strategies to enable the adoption of animal biotechnology to sustainably improve global food safety and security.

The ability to generate transgenic animals has existed for over 30 years, and from those early days many predicted that the technology would have beneficial applications in agriculture. Numerous transgenic agricultural animals now exist, however to date only one product from a transgenic animal has been approved for the food chain, due in part to cumbersome regulations. Recently, new techniques such as precision breeding have emerged, which enables the introduction of desired traits without the use of transgenes.

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease.

Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos.

Improving livestock for agriculture - technological progress from random transgenesis to precision genome editing heralds a new era.

Humans have a long history in shaping the genetic makeup of livestock to optimize production and meet growing human demands for food and other animal products. Until recently, this has only been possible through traditional breeding and selection, which is a painstakingly slow process of accumulating incremental gains over a long period. The development of transgenic livestock technology offers a more direct approach with the possibility for making genetic improvements with greater impact and within a single generation.

Coupled solid phase extraction and microparticle-based stability and purity-indicating immunosensor for the determination of recombinant human myelin basic protein in transgenic milk.

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk.

Transient JMJD2B-mediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos.

Correct reprogramming of epigenetic marks in the donor nuclei is crucial for successful cloning by nuclear transfer. Specific epigenetic modifications, such as repressive histone lysine methylation marks, are known to be very stable and difficult to reprogram. The discovery of histone lysine demethylases has opened up opportunities to study the effects of removing repressive histone lysine methylation marks in donor cells prior to nuclear transfer.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine.

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk.

Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG.

Primary transgenic bovine cells and their rejuvenated cloned equivalents show transgene-specific epigenetic differences.

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential.