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. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. METHODS: In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. KEY RESULTS: Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. CONCLUSIONS: Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. IMPLICATIONS: The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions.

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). The subjects were weighed, scored and subjected to behavioural tests. After reaching a clinical score of 3, various treatments were given to corresponding EAE-induced and non-induced groups including rhMBP, RTX, empty nanoparticle prepared by poly (lactide-co-glycolide) (PLGA) or the prepared nanoformulation (Nano-rhMBP-RTX). At the end of the study, biochemical parameters were also determined as interferon-γ (IFN-γ), myeloperoxidase (MPO), interleukin-10 (IL-10), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-kB), brain derived neurotrophic factor (BDNF), 2', 3' cyclic nucleotide 3' phosphodiesterase (CNP) and transforming growth factor beta (TGF-ß) along with some histopathological analyses. Results: The results of the Nano-rhMBP-RTX group showed promising outcomes in terms of reducing the clinical scores, improving the behavioural responses associated with improved histopathological findings. Elevation in the levels of IL-4, CNP and TGF-ß was also noticed along with marked decline in the levels of NF-kB and TNF-α. Conclusion: Nano-rhMBP-RTX treated group ameliorated the adverse effects induced in the EAE model. The effectiveness of this formulation was demonstrated by the normalization of EAE-induced behavioral changes and aberrant levels of specific biochemical markers as well as reduced damage of hippocampal tissues and retaining higher levels of myelination.

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. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

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., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

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. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10 g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for 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. Herein we report that gCetuximab has similar bioactivity and pharamacokinetic properties compared with the commercial product produced in mammalian cell culture. In particular both forms have very similar efficacy in a HT29 colorectal cancer xenograft model alone or when conjugated to the toxin MMAE. This also demonstrates that the gCetuximab will be a viable vehicle for antibody drug conjugate based therapies. Taken together, this shows that the goat milk monoclonal antibody production system is an effective way of producing a biosimilar form of cetuximab.

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. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.

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. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves. The third calf, a healthy female, had in addition to the expected nine bp deletion (81%), alleles with an in frame 21 bp deletion (<17%) at the target site. While her milk was free of any mature BLG, we detected low levels of a BLG variant derived from the minor deletion allele. This confirmed that the nine bp deletion genotype completely knocks out production of BLG. In addition, we showed that the LGB knockout animals are free of any TALEN-mediated off-target mutations or vector integration events using an unbiased whole genome analysis. Our study demonstrates the feasibility of generating precisely biallelically edited cattle by zygote-mediated editing for the safe production of hypoallergenic milk.

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. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications.

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. Cloned fetuses from aborted pregnancies and a cloned live calf that died shortly after birth were examined and confirmed to all possess tails. Hence, the observed phenotype of Daisy's lacking tail is not due to the introduced transgene or a mutation present in the cell that was used for her production. Rather, the missing tail has most likely arisen from an epigenetic reprogramming error during development.

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). Upon inducing Kdm4b, H3K9/36me3 levels significantly decreased compared to non-induced controls. Concurrently, H3K9me1 levels significantly increased, while H3K9me2 and H3K27me3 remained unchanged. The global transcriptional impact of Kdm4b-mediated reduction in H3K9/36me3 levels was examined by comparative microarray analysis and mRNA-sequencing of three independent transgenic MEF lines. We identified several commonly up-regulated targets, including the heterochromatin-associated zinc finger protein 37 and full-length endogenous retrovirus repeat elements. Following optimized zona-free somatic nuclear transfer, reduced H3K9/36me3 levels were restored within hours. Nevertheless, hypo-methylated Kdm4b MEF donors reprogrammed six-fold better into cloned blastocysts than non-induced donors. They also reprogrammed nine-fold better into induced pluripotent stem cells that gave rise to teratomas and chimeras. In summary, we firmly established H3K9/36me3 as a major roadblock to somatic cell reprogramming and identified transcriptional targets of derestricted chromatin that could contribute towards improving this process in mouse.

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

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. Here, the activity of free and controlled release rhMBP poly(ε-caprolactone) nanoparticles (NPs), as a therapeutic vaccine against multiple sclerosis (MS) was demonstrated in experimental autoimmune encephalomyelitis (EAE) animal model. Following optimization of nanoformulation, discrete spherical, rough-surfaced rhMBP NPs with high entrapment efficiency and controlled release pattern were obtained. Results indicated that rhMBP was loaded into and electrostatically adsorbed onto the surface of NPs. Subcutaneous administration of free or rhMBP NPs before EAE-induction reduced the average behavioral score in EAE mice and showed only mild histological alterations and preservation of myelin sheath, with rhMBP NPs showing increased protection. Moreover, analysis of inflammatory cytokines (IFN-γ and IL-10) in mice brains revealed that pretreatment with free or rhMBP NPs significantly protected against induced inflammation. IN CONCLUSION: i) rhMBP ameliorated EAE symptoms in EAE animal model, ii) nanoformulation significantly enhanced efficacy of rhMBP as a therapeutic vaccine and iii) clinical investigations are required to demonstrate the activity of rhMBP NPs as a therapeutic vaccine for MS.

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

We have previously generated transgenic cattle with additional copies of bovine ß- 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 ß-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. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional ß- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics.

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. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

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. The rapidly growing human population, environmental degradation, and concerns related to zoonotic and pandemic diseases have increased pressure on the animal agriculture sector to provide a safe, secure and sustainable food supply. There is a clear need to adopt transgenic technologies as well as new methods such as gene editing and precision breeding to meet these challenges and the rising demand for animal products. To achieve this goal, cooperation, education, and communication between multiple stakeholders-including scientists, industry, farmers, governments, trade organizations, NGOs and the public-is necessary. This report is the culmination of concepts first discussed at an OECD sponsored conference and aims to identify the main barriers to the adoption of animal biotechnology, tactics for navigating those barriers, strategies to improve public perception and trust, as well as industry engagement, and actions for governments and trade organizations including the OECD to harmonize regulations and trade agreements. Specifically, the report focuses on animal biotechnologies that are intended to improve breeding and genetics and currently are not routinely used in commercial animal agriculture. We put forward recommendations on how scientists, regulators, and trade organizations can work together to ensure that the potential benefits of animal biotechnology can be realized to meet the future needs of agriculture to feed the world.

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. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.

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. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

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. However, initially the technology was hampered by technical difficulties and limitations, which have now largely been overcome by progressive improvements over the past 30 years. Particularly, the advent of genome editing in combination with homologous recombination has added a new level of efficiency and precision that holds much promise for the genetic improvement of livestock using the increasing knowledge of the phenotypic impact of genetic sequence variants. So far not a single line of transgenic livestock has gained approval for commercialization. The step change to genome-edited livestock with precise sequence changes may accelerate the path to market, provided applications of this new technology for agriculture can deliver, in addition to economic incentives for producers, also compelling benefits for animals, consumers, and the environment.

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. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04 ng mL(-1)≈0.3 n mol L(-1)) and for quantitative determination over a wide concentration range (10.00-10,000.00 ng mL(-1)). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry.

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. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.