Over-expression of CD163, CD169, and CD151 is not sufficient to improve the susceptibility to porcine reproductive and respiratory syndrome virus infection in transgenic mice

Porcine reproductive and respiratory syndrome virus(PRRSV)is a major pathogen that causes reproductive failure and respiratory disease in pigs,resulting in devastating economic losses worldwide[1].Porcine alveolar macrophages(PAMs)are the primary target cells of PRRSV[2],and the putative receptors,including CD163,CD169,and CD151,play key roles during infection[3–6].However,the understanding of PRRSV infection and pathogenesis is

Advances in genetic engineering of domestic animals

Global population will increase to over nine billion by 2050 with the doubling in demand for meat and milk. To overcome this challenge, it is necessary to breed highly ef ficient and productive livestock. Furthermore,livestock are also excellent models for human diseases and ideal bioreactors to produce pharmaceutical proteins.Thus, genetic engineering of domestic animals presents a critical and valuable tool to address these agricultural and biomedical applications. Overall, genetic engineering has evolved through three stages in history: transgenesis, gene targeting, and gene editing. Since the birth of the first transgenic pig, genetic engineering in livestock has been advancing slowly due to inherent technical limitations. A major breakthrough has been the advent of somatic cell nuclear transfer, which, for the first time, provided the technical ability to produce site-specific genome-modi fied domestic animals. However, the low efficiency of gene targeting events in somatic cells prohibits its wide use in agricultural and biomedical applications. Recently, rapid progress in tools and methods of genome engineering has been made, allowing genetic editing from mutation of a single base pair to the deletion of entire chromosomes.Here, we review the major advances of genetic engineering in domestic animals with emphasis placed on the introduction of latest designer nucleases.

Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

In this study,we introduced the bovine immunoglobulinμheavy-chain gene(the orphaned gene on BTA11)into mouse germline cells.Bovine IgM was highly expressed in selected transgenic lines,and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain(IgH)genes in these lines.The forced expression of bovine IgM resulted in reduced numbers of pro-and pre-B cells but increased the number of immature B cells in the transgenic mice.Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen,but most of the cells are arrested at the T1 transitional B cell stage,leading to a significantly lower number of T2 transitional and mature B cells in the spleen.Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased,the IgA levels were slightly increased compared to the WT mice.The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM,suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells.These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3(CDR3)sequences in their VH repertoire and Vκrepertoire but exhibited an increased frequency of unique CDR3 in their Vλrepertoire.Compared to the WT mice,the transgenic mice had a significantly higher percentage of mouse IgMexpressing B cells that expressedλchains.Finally,we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.

Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins.While numerous physiological functions have been described for lactoferrin,the mechanisms underlying these functions are not clear.To further study the functions and mechanisms of lactoferrin,we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression.Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24%(7/29)by injecting the plasmid pX330,expressing a small guide RNA and human codonoptimized SpCas9,into fertilized eggs of mice.Plasmid integration and off-targeting of pX330 were not detected.These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76
12 to 159
28 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.