Biochemistry, Genetics and Molecular Biology › Genetics

Animal Genetics and Reproduction

Works Count: 86361

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Description

This cluster of papers explores the advancements in genetic modification and biopharmaceutical production using transgenic animals. It covers topics such as germ cell transfer, livestock biotechnology, gene expression, animal welfare, and the application of transgenesis in aquaculture. The research also delves into genomic integration and the use of embryonic stem cells in transgenic animal production.

Keywords

Transgenic Animals; Genetic Modification; Biopharmaceutical Production; Germ Cell Transfer; Livestock Biotechnology; Gene Expression; Animal Welfare; Aquaculture; Genomic Integration; Embryonic Stem Cells

Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis.

1991-12-01

Jeanne Keffer , Lesley Probert , Haris Cazlaris +4 more

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In situ hybridization; a practical approach

1993-09-01

D. G. Wilkinson

Genetic variants and strains of the laboratory mouse

1990-11-01

M. F. Lyon , Antony G. Searle

Rules and guidelines for gene nomenclature, Mary F.Lyon catalogue of mutant genes and polymorphic loci, Margaret C. Green retroviral and cancer-related genes, Christine A.Kozak linkage map, Muriel T.Davisson and Thomas … Rules and guidelines for gene nomenclature, Mary F.Lyon catalogue of mutant genes and polymorphic loci, Margaret C. Green retroviral and cancer-related genes, Christine A.Kozak linkage map, Muriel T.Davisson and Thomas H.Roderick map of the t-complex, Anna-Maria Frischauf recombination percentages and chromosomal assignments, Muriel T.Davisson et al linkage and synteny homologies in mouse and man, Joseph H.Nadeau and Andrew H.Reiner DNA polymorphisms, Rosemary W.Elliott highly repeated DNA families in the genome of mus-musculus, Nicholas D.Hastie rules for nomenclature of chromosome anomalies, Mary F.Lyon standard idiogram, Edward P.Evans standard G-banded karyotype, Edward P.Evans standard karyotype of early replicating bands, Imre E. Somssich and Horst Hameister numerical variants and structural rearragements, Anthony G.Searle centromeric heterochromatin variants, Muriel T.Davisson nucleolus organizer regions, Muriel T.Davisson map of reciprocal translocations, inversions and insertions, Anthony G.Searle and Colin V.Beechey map of inversions, Thomas H. Roderick et al rules for nomenclature of inbred strains, Mary F.Lyon inbred strains of mice, Michael F.W.Festing the wild house mouse and its relatives, Francois Bonhomme and Jean-Louis Guenet strain distribution of polymorphic variants, Thomas H.Roderick and John H.Guidi recombinant inbred strains, Benjamin A.Taylor immunologically important loci, Jan Klein mutant genes and biochemical loci, Priscilla W.Lane and Mary F.Lyon subline codes for holders and producers, Dorothy D.Greenhouse.

Cell biology :a laboratory handbook.

1994-01-01

Julio E. Celis

Gene therapy - promises, problems and prospects

1997-09-18

Inder M. Verma , Nikunj V. Somia

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Generalized lacZ expression with the ROSA26 Cre reporter strain

1999-01-01

Philippe Soriano

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

1988-02-01

Cathy A. Finlay , Philip W. Hinds , Tse‐Hua Tan +3 more

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino … The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.

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A genetic screen for mutations affecting embryogenesis in zebrafish.

1996-12-01

Wolfgang Driever , Lilianna Solnica‐Krezel , Alexander F. Schier +7 more

Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a … Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pigmentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.

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Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice

1988-08-01

Christopher C. Goodnow , J. Crosbie , Stephen Adelstein +7 more

An Air-Drying Method for Chromosome Preparations from Mouse Eggs

1966-01-01

A Tarkowski

The eggs, after being washed out from the genital tract, are put into hypotonic (1%) sodium citrate and left to stand at room temperature for 5–15 minutes. The duration of … The eggs, after being washed out from the genital tract, are put into hypotonic (1%) sodium citrate and left to stand at room temperature for 5–15 minutes. The duration of treatment is not very critical, but later stages, especially blastocysts, need longer treatment. A microdrop of this solution together with the eggs is placed on a grease-free slide. A few drops of acetic alcohol (3 parts of absolute ethyl alcohol, 1 part of glacial acetic acid) are immediately expelled from another pipette, whose tip is brought just over the microdrop containing eggs. The optimal number of drops of fixative has been found to be three in the case of eggs from the one cell to eight cell stage and five or even more in the case of blastocysts. These indications were calculated for drops measuring approximately 0.02 ml in volume. Air-dried preparations are stained in lactic-acetic-orcein or in 2% aqueous solution of toluidine blue. The method can be applied to all stages from diakinesis of the first meiotic division to blastocyst. It enables one to obtain excellent scattering of nuclei and metaphase plates and good spreading of chromosomes. The remnants of cytoplasm are negligible.

Sheep cloned by nuclear transfer from a cultured cell line

1996-03-01

Keith Campbell , J. McWhir , William A. Ritchie +1 more

GERM-LINE TRANSFORMATION OF MICE

1986-12-01

Richard D. Palmiter , Ralph L. Brinster

Orthologs and paralogs are two fundamentally different types of homologous genes that evolved, respectively, by vertical descent from a single ancestral gene and by duplication. Orthology and paralogy are key … Orthologs and paralogs are two fundamentally different types of homologous genes that evolved, respectively, by vertical descent from a single ancestral gene and by duplication. Orthology and paralogy are key concepts of evolutionary genomics. A ...Read More

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A series of normal stages in the development of the chick embryo

1992-12-01

Viktor Hamburger , Howard L. Hamilton

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COLLAGENOLYTIC ACTIVITY IN AMPHIBIAN TISSUES: A TISSUE CULTURE ASSAY

1962-06-01

Jerome Gross , Charles M. Lapière

Gene expression variation is shaped by both genetic and environmental effects, yet these two factors are rarely considered together in the context of adaptive evolution. We studied environmental influences on … Gene expression variation is shaped by both genetic and environmental effects, yet these two factors are rarely considered together in the context of adaptive evolution. We studied environmental influences on gene regulatory evolution in ...Changes in gene expression are thought to play a major role in adaptive evolution. While it is known that gene expression is highly sensitive to the environment, very few studies have determined the influence of genetic and environmental effects on ...

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Cloned Transgenic Calves Produced from Nonquiescent Fetal Fibroblasts

1998-05-22

José B. Cibelli , Steven L. Stice , Paul J. Golueke +5 more

An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a … An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking

1995-02-01

Yao-Guang Liu , Robert F. Whittier

Prostate cancer in a transgenic mouse.

1995-04-11

Norman M. Greenberg , Francesco J. DeMayo , Milton J. Finegold +7 more

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop … Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

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Early development of Xenopus laevis : a laboratory manual

2000-01-01

Hazel Sive , Robert M. Grainger , Richard M. Harland

Preface Acknowledgements Chapter 1: Introduction Chapter 2: Morphology of Xenopus Embryos and Adults Chapter 3: Manipulating Gene Expression in Xenopus Chapter 4: Equipment for Embryo Experiments Chapter 5: Obtaining Embryos … Preface Acknowledgements Chapter 1: Introduction Chapter 2: Morphology of Xenopus Embryos and Adults Chapter 3: Manipulating Gene Expression in Xenopus Chapter 4: Equipment for Embryo Experiments Chapter 5: Obtaining Embryos Chapter 6: Preparing Embryos for Manipulation Chapter 7: Embryo Perturbations Chapter 8: Microinjection Chapter 9: Fate Mapping and Lineage Labeling Chapter 10: Microdissection Chapter 11: Transgenesis in Xenopus Embryos Chapter 12: Immunocytochemistry Chapter 13: Whole-mount In situ Hybridization Chapter 14: Histology Appendix 1: Culture Media and Solutions Appendix 2: Timing of Development of Xenopus laevis Embryos and Temperature Dependence Appendix 3: Nucleic Acid Methods Appendix 4: Cautions Appendix 5: Suppliers Appendix 6: Trademarks Index

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

1996-06-11

Luda Diatchenko , Yun‐Fai Chris Lau , A. Patricia Campbell +7 more

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique … A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

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Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

1991-09-01

G Friedrich , Philippe Soriano

A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both … A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.

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Spermatogenesis following male germ-cell transplantation.

1994-11-22

Ralph L. Brinster , James W. Zimmermann

In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. … In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.

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Germline transmission of donor haplotype following spermatogonial transplantation.

1994-11-22

Ralph L. Brinster , Mary R. Avarbock

Spermatogenesis is a complex, highly organized, very efficient process that is based upon the capacity of stem cell spermatogonia simultaneously to undergo self-renewal and to provide progeny that differentiate into … Spermatogenesis is a complex, highly organized, very efficient process that is based upon the capacity of stem cell spermatogonia simultaneously to undergo self-renewal and to provide progeny that differentiate into mature spermatozoa. We report here that testis-derived cells transplanted into the testis of an infertile mouse will colonize seminiferous tubules and initiate spermatogenesis in > 70% of recipients. Testis-derived cells from newborn mice were less effective in colonizing recipient testes than cells from 5- to 15- or 21- to 28-day-old mice. Increasing the number of Sertoli cells in the donor cell population did not increase the efficiency of colonization. Unmodified embryonic stem cells were not able to substitute for testis-derived cells in colonizing testes but instead formed tumors in syngeneic as well as nonsyngeneic hosts. Finally, with recipients that maintained endogenous spermatogenesis, testis cell transplantation yielded mice in which up to 80% of progeny were sired by donor-derived spermatozoa. The technique of spermatogonial cell transplantation should provide a means to generate germline modifications in a variety of species following development of spermatogonial culture techniques and should have additional applications in biology, medicine, and agriculture.

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Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products

1991-01-01

Douglas A. Marchuk , Mitchell L. Drumm , Ann M. Saulino +1 more

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Animal cytology and evolution

1980-02-01

M. J. D. White

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Rapid and efficient site-specific mutagenesis without phenotypic selection.

1985-01-01

Thomas A. Kunkel

Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products … Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

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Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.

1990-06-01

Seth G. N. Grant , Joel Jessee , F R Bloom +1 more

Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the … Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS). Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression. Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E. coli strains carrying mutations in two of the MDRS loci, mcrA and mcrB. In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E. coli that carry mutations in all known MDRS activities. Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes. These multiply deficient E. coli strains will also facilitate the molecular cloning of modified chromosomal DNA.

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Establishment in culture of pluripotential cells from mouse embryos

1981-07-01

Martin Evans , M. H. Kaufman

High-resolution in situ hybridization to whole-mount zebrafish embryos

2007-12-20

Christine Thisse , Bernard Thisse

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

1996-06-11

Merja Lakso , José G. Pichel , James Gorman +5 more

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. … We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

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Functional messenger RNAs are produced by SP6in vitrotranscription of cloned cDNAs

1984-01-01

Paul A. Krieg , Douglas A. Melton

We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these … We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these synthetic RNAs function efficiently as messenger RNAs. We confirm that a 5′ cap on the mRNA is essential for translation in injected oocytes and show that most of the 3′ flanking region, including the poly A tail, can be deleted without the abolition of protein synthesis. The method of mRNA synthesis involves invitro transcription of cDNAs which have been cloned into SP6 vectors (described in the accompanying paper). This method enables one to produce large amounts of mRNA and consequently protein from any cDNA clone.

Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

2000-01-14

Xudong Ye , Salim Al‐Babili , Andreas Klöti +4 more

Rice ( Oryza sativa ), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible … Rice ( Oryza sativa ), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

The atlas of mouse development

1993-01-01

Rosa Beddington

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

1993-09-01

Jane C. Burns , T Friedmann , Wolfgang Driever +2 more

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived … The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.

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Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division

1983-06-01

Tom Evans , Eric T. Rosenthal , Jim Youngblom +2 more

Dramatic growth of mice that develop from eggs microinjected with metallothionein–growth hormone fusion genes

1982-12-01

Richard D. Palmiter , Ralph L. Brinster , Robert E. Hammer +4 more

Ligation-independent cloning of PCR products (LIC-PCR)

1990-01-01

Charalampos Aslanidis , Pieter J. de Jong

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a … A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5′-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3′-ends. The 3′-terminal sequence can be removed by the action of the (3′→5′) exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5′-extendlng single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences In the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.

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Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

1988-12-01

Michael A. Frohman , Michael Dush , Gail R. Martin

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated … We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

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Genome sequence, comparative analysis and haplotype structure of the domestic dog

2005-12-01

Kerstin Lindblad‐Toh , Claire M. Wade , Tarjei S. Mikkelsen +7 more

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The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio

1996-12-01

Pascal Haffter , Michael Granato , Michael Brand +7 more

ABSTRACT In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and … ABSTRACT In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereo-microscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

A series of normal stages in the development of the chick embryo

1951-01-01

Viktor Hamburger , Howard L. Hamilton

Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction

1997-02-01

Kirk M. Ririe , Randy Rasmussen , Carl T. Wittwer

Biology of the Laboratory Mouse

1967-01-01

W. E. Heston

Journal Article Biology of the Laboratory Mouse Get access W. E. HESTON W. E. HESTON National Cancer Institute, National Institutes of HealthBethesda, Maryland 20014 Search for other works by this … Journal Article Biology of the Laboratory Mouse Get access W. E. HESTON W. E. HESTON National Cancer Institute, National Institutes of HealthBethesda, Maryland 20014 Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Heredity, Volume 58, Issue 1, January 1967, Page 16, https://doi.org/10.1093/oxfordjournals.jhered.a107528 Published: 01 January 1967

The Journal of Animal Science: 1988-1990

1992-12-01

F. N. Owens

Current topics in developmental biology

1998-12-01

In addition he argues strongly for the inclusion of Homo habilis within the genus Australo- pithecus, thus regarding all the African sub-Saharan Lower Pleistocene hominids as belonging to one genus … In addition he argues strongly for the inclusion of Homo habilis within the genus Australo- pithecus, thus regarding all the African sub-Saharan Lower Pleistocene hominids as belonging to one genus ancestral to man-a viewpoint widely but not universally accepted.At least one admitted authority, Professor John Robinson, believes that the large and small australopithecines are generically distinct, and that there is evidence of the larger form outside the continent of Africa.

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Positional cloning of the mouse obese gene and its human homologue

1995-03-01

Yiying Zhang , Ricardo Proenca , Margherita Maffei +3 more

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The "megaprimer" method of site-directed mutagenesis.

1990-04-01

Gobinda Sarkar , S S Sommer

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In … We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.

RP267 Assessment of the Application for the Feed Additive Pediococcus Pentosaceus IMI507024 for All Animal Species

2025-06-25

| FSA research and evidence.

The FSA/ FSS have undertaken a safety assessment of application RP 267 for the use of Pediococcus pentosaceus IMI507024 as a feed additive for all animal species, from ALL-TECHNOLOGY (Ireland) … The FSA/ FSS have undertaken a safety assessment of application RP 267 for the use of Pediococcus pentosaceus IMI507024 as a feed additive for all animal species, from ALL-TECHNOLOGY (Ireland) LIMITED. FSA/FSS has reviewed the EFSA opinion (EFSA Journal 2021;19(7):6701) and confirm that it is adequate for UK considerations and therefore a full safety assessment of this application was not performed by FSA and FSS. In line with the principles for making use of EFSA opinions in their decision making on regulated products, the FSA/FSS opinion is that the conclusions of the EFSA opinion are valid for the UK and therefore Pediococcus pentosaceus IMI507024, as described in this application, is safe and is not liable to have an adverse effect on the target species, worker safety, environmental safety and human health at the intended concentrations of use.

RP270 Assessment of the Application for the Feed Additive Pediococcus Pentosaceus IMI507025 for All Animal Species

2025-06-25

| FSA research and evidence.

The FSA/ FSS have undertaken a safety assessment of application RP 270 for the use of Pediococcus pentosaceus IMI507025 as a feed additive for all animal species, from ALL-TECHNOLOGY (Ireland) … The FSA/ FSS have undertaken a safety assessment of application RP 270 for the use of Pediococcus pentosaceus IMI507025 as a feed additive for all animal species, from ALL-TECHNOLOGY (Ireland) LIMITED. FSA/FSS has reviewed the EFSA opinion (EFSA Journal 2021;19(7):6702) and confirm that it is adequate for UK considerations and therefore a full safety assessment of this application was not performed by FSA and FSS. In line with the principles for making use of EFSA opinions in their decision making on regulated products, the FSA/FSS opinion is that the conclusions of the EFSA opinion are valid for the UK and therefore Pediococcus pentosaceus IMI507025, as described in this application, is safe and is not liable to have an adverse effect on the target species, worker safety, environmental safety and human health at the intended concentrations of use.

An atlas of cell type specific regulatory effects in cattle

2025-06-24

Houcheng Li , Huicong Zhang , Pengju Zhao +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Understanding the genetic and molecular architecture of complex traits and artificial selection is crucial for advancing sustainable precision breeding in cattle and other livestock. Yet, how genetic variation affects cellular … Understanding the genetic and molecular architecture of complex traits and artificial selection is crucial for advancing sustainable precision breeding in cattle and other livestock. Yet, how genetic variation affects cellular gene expression remains elusive in cattle. Here, by integrating 8,866 bulk RNA-seq samples and 999,192 single cells of 81 cell types in 22 bovine tissues, we presented a comprehensive atlas of regulatory variants at the cell type resolution in cattle. By colocalizing with bulk-tissue expression quantitative trait loci (beQTL), we detected 57,043 novel cell-type stratified eQTL and cell-type/state interaction eQTL in 18,153 genes, which also exhibited a stronger tissue/cell-type specificity than beQTL. By examining genome-wide associations (GWAS) of 44 complex traits, these cell-resolved eQTL were colocalized with 505 (24%) additional GWAS loci compared to beQTL. Through integrating this resource with selection signatures between dairy and beef cattle, we provided tissue/cell-specific regulatory insights into cattle breeding. Overall, the current atlas of cell-type-specific regulatory variants will serve as an invaluable resource for cattle genomics and selective breeding.

ScienceAdviser: Orcas get by with a little kelp from their friends

2025-06-24

| AAAS Articles DO Group

Exploring MAPK and mTOR Pathways in Feline Thyroid Tumors

2025-06-24

Alexandra Monteiro , Tiago Bordeira Gaspar , Inês Borges +5 more | Veterinary Sciences

Thyroid tumors are common in humans and cats, occurring most commonly as benign lesions, whereas thyroid carcinoma is barely detected in both species. Determining the mutational status of MAPK-related genes … Thyroid tumors are common in humans and cats, occurring most commonly as benign lesions, whereas thyroid carcinoma is barely detected in both species. Determining the mutational status of MAPK-related genes (BRAF, NRAS, HRAS, and KRAS) and the activation status of MAPK and mTOR pathways is crucial for establishing the diagnosis, treatment, and prognosis of human patients. So far, the role of such players in feline thyroid tumorigenesis remains underexplored. This study aims to elucidate the presence and implications of potential shared molecular mechanisms between human and feline thyroid tumors. Fifteen formalin-fixed paraffin-embedded feline thyroid epithelial tumors (four tumors with atypia and 11 with no atypia) were collected to perform mutational and immunohistochemical analyses. Sanger sequencing targeting human homologous hotspots revealed no mutations in BRAF (human codon 600) or RAS (human codon 61) regions. A KRAS missense mutation (p.Gln232His) was identified in two tumors with no atypia of follicular pattern (2/15, 13%). Regardless of the mutational status, pERK (Thr202/Ty204) was immuno-expressed in 10/11 (91%), pS6 (Ser235/236) in 100%, and pAKT (Ser473) in 8/11 (73%) of the tumors with no atypia. The expression patterns of pERK, pS6, and pAKT and their associations with clinical-pathological features seem to mirror the progression dynamics observed in human thyroid tumorigenesis. pAKT expression was associated with the presence of multiple tumor foci within the same thyroid lobe, suggesting its potential as a marker of aggressiveness in feline thyroid tumors. This study introduces cats as potential animal models for human thyroid tumorigenesis, with further research required to confirm such potential.

Transcripts of COL4A2, IRG1, MMP12 genes as potential biomarkers of bull sperm quality

2025-06-24

Olga Y. Barkova , D. A. Starikova , I. V. Chistyakova +2 more | Agrarian science

Relevance . The aim of this work is to validate three sperm transcripts of candidate genes COL4A2, IRG1, MMP12 obtained using RNA-Seq and correlation analysis of their expression levels with … Relevance . The aim of this work is to validate three sperm transcripts of candidate genes COL4A2, IRG1, MMP12 obtained using RNA-Seq and correlation analysis of their expression levels with high and low sperm quality indicators of Holstein bulls. Methods . Eighteen samples of cryopreserved sperm ejaculate were studied. The relative expression of candidate genes COL4A2, IRG1, MMP12 was analyzed, and rank correlation was analyzed using Spearman’s test. Results . The results of the study showed that the relative expression of the studied genes COL4A2, IRG1, MMP12 was predominantly lower in spermatozoa with low productivity, which confirms the results of a complete transcriptome analysis, where reduced differential expression was also observed in the group of bulls with poor productivity. Statistically significant differences in the levels of expression of total sperm RNA of the studied genes were obtained for all genes in both groups of bulls. Correlation analysis revealed a high dependence between the expression level of the studied genes COL4A2, IRG1, MMP12 and significant indicators of sperm quality. Transcripts of the genes COL4A2, IRG1 showed a high significant negative correlation with sperm motility, the content of live and normal cells and the average Ca 2 + content. A significant positive correlation was observed for undesirable traits, such as the content of dead and defective cells. The gene MMP12 transcript had the opposite effect in comparison with the genes IRG1 and COL4A2 . The expression level of the MMP12 gene showed a significantly high correlation with motility.

Chicken Primordial Germ Cell Surface Marker

2025-06-24

Tamara J. Gough , Terry G. Wise , Matthew P. Bruce +3 more | Animals

The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens … The creation of transgenic chickens holds significant promise for the agricultural and biotechnological sectors, offering potential improvements in disease resistance and production efficiency. The preferred method for generating gene-edited chickens involves the genetic manipulation of primordial germ cells (PGCs), making the identification and isolation of these cells a growing focus of research. PGCs are the precursors to sperm and oocytes, responsible for transmitting genetic material to the next generation. In humans, PGCs are characterized by their large size, round nuclei, and refractive lipids in the cytoplasm, and can be identified using periodic acid–Schiff (PAS) staining and the surface marker stage-specific embryonic antigen 1 (SSEA1). Similarly, chicken PGCs express SSEA1, but their most specific marker is the chicken vasa homologue (CVH), the avian equivalent of the RNA-binding factor gene vasa. However, SSEA1, along with other known surface markers, does not bind to all PGCs or lacks specificity, while CVH, although highly specific to PGCs, is intracellular and unsuitable for isolating viable cells. This study aims to develop an antibody targeting a PGC surface marker with the same specificity as CVH. Despite the importance of identifying surface markers for PGC characterization, to date, such reagents are limited. To address this, whole chicken PGCs were injected into mice, leading to the generation of a panel of monoclonal antibodies. One antibody was found to bind cultured chicken PGCs and showed reduced expression upon differentiation with retinoic acid, indicating its specificity to PGCs. Immunoprecipitation followed by mass spectrometry identified the antigen as myosin heavy chain-like (MYH9) protein. The antibody, αMYH9, was further characterized and shown to bind circulating PGCs and embryonic gonadal PGCs (Hamburger Hamilton (H-H) stage 30, embryonic day 6.5–7). Whilst our primary aim was to determine the binding to PGCs, further investigation is required to determine potential binding to somatic cells. In conclusion, this study provides the characterization of a surface marker for chicken PGCs, with significant implications for advancements in avian genetic preservation, agriculture, and biotechnology.

Establishment of two continuous adult stem cell lines from muscle and adipose tissues of rainbow trout (Oncorhynchus mykiss) for marbled fish meat production

2025-06-23

Yaru Li , Xue Ting , Song Wang +4 more | Aquaculture Reports

CONSUMERS’ KNOWLEDGE AND VIEWS ON PRODUCTS ORIGINATING FROM NATIVE ANIMAL BREEDS

2025-06-23

Aleksandra Haska , E. Martyniuk | Folia Pomeranae Universitatis Technologiae Stetinensis Agricultura Alimentaria Piscaria et Zootechnica

One of the best strategies for ensuring the long-term survival of native breeds is to promote their products which may increase the profitability of their use. The aim of this … One of the best strategies for ensuring the long-term survival of native breeds is to promote their products which may increase the profitability of their use. The aim of this study was to assess consumers’ knowledge of food products from raw materials obtained from Polish native breeds and to analyse their perception of the market of niche products. The study examined customers’ motivation when purchasing animal origin niche products, as well as their willingness to pay higher prices for such products. In total, 255 respondents participated in the survey based on an anonymous questionnaire. Statistically significant effects of socio-demographic factors such as gender, age group, education level, and income levels, as well as understanding of the term “native or local breed”, were observed for consumers’ knowledge about animal origin niche products, their labels, and certification, as well as their market. All of the analysed factors had a significant impact on the willingness to pay a higher price for niche products originating from native breeds. The highest level of knowledge and interest in buying such niche products was observed in the case of middle-aged, well-educated, high-income consumers, especially women. The study showed that it is necessary to undertake well-organized and targeted education and information activities, as the lack of consumer's knowledge seems to be the main barrier to enhancing demand for products from local breeds. The higher level of consumer acceptance will support the development of a niche product market, which could enhance profitability of utilization of Polish native breeds, and thus, provide the potential to support their long-term conservation

Marine Biotechnology

2025-06-23

Anjana K. Vala , Dushyant R. Dudhagara | CRC Press eBooks

Omics Translation Marine-Derived Pharmaceuticals for the Blue Bio-economy

2025-06-23

Parthkumar Prajapati , Bhavin Parekh , Rimpal Bhanderi +2 more | CRC Press eBooks

Blastocyst complementation: current progress and future directions in xenogeneic organogenesis

2025-06-23

Paula Barlabé , Xabier L. Aranguren , Giulia Coppiello | Stem Cell Research & Therapy

The generation of organs derived from pluripotent stem cells can be achieved in vivo through the blastocyst complementation technique. This method is based on the introduction of pluripotent stem cells … The generation of organs derived from pluripotent stem cells can be achieved in vivo through the blastocyst complementation technique. This method is based on the introduction of pluripotent stem cells into organogenesis-disabled pre-implantation embryos, where environmental signals instruct donor cells to colonize the vacant niche and to develop into the missing organ. When applied interspecies, this approach has the potential to produce human organs in genetically engineered livestock, offering a promising solution to the global transplants' shortage crisis. In this review, we summarize the current progress in blastocyst complementation research and highlight the key challenges that must be addressed to advance this field.

Shell color and growth traits share the same major locus in Cyclina sinensis

2025-06-20

Hengde Li , Xiao-Dong Ma , Yaqun Zhang +4 more | Aquaculture Reports

Genotype-Based Housing as a Potential Confounder in Studies Using Transgenic Mouse Models—Insight from the A53T Mouse Model of Parkinson’s Disease

2025-06-19

Olga Dubljević , Miodrag Dragoj , Milica Potrebić +3 more | Biomedicines

Background/Objectives: Environmental factors, including the differences in genotype-based housing (GbH), can act as confounding variables in studies using transgenic mouse models, potentially influencing experimental outcomes and limiting their reproducibility and … Background/Objectives: Environmental factors, including the differences in genotype-based housing (GbH), can act as confounding variables in studies using transgenic mouse models, potentially influencing experimental outcomes and limiting their reproducibility and translational value. Despite the widespread use of transgenic models in preclinical studies, the extent to which housing conditions can affect the behavioral and molecular parameters of interest remains poorly understood. This study aims to investigate how different GbH conditions influence visuo-spatial memory and gene expression in the A53T mouse model (JAX006823) of Parkinson's disease (PD) during the pre-motor phase. Methods: A53T+ transgenic male mice and their non-transgenic littermates (A53T-) were housed in either mixed-genotype (MGH) or single-genotype (SGH) environments from postnatal day (PND) 30, with C57BL/6J mice serving as the controls. A behavioral assessment using the Novel Object Recognition and Object Location Tests was conducted at PND 180, followed by a qPCR analysis of Iba1, Gfapα, Bdnf, Tnfα, Il-1β, and Il-6 expression in the medial prefrontal cortex and the hippocampus. Results: The variations in GbH influenced behavior and mRNA expression differently in the A53T+ and A53T- animals. Specifically, the A53T- mice in SGH environments displayed behavioral and molecular profiles similar to the C57BL/6J controls, while the same was not evident in the MGH environments. In the A53T+ mice, the mRNA expression of Iba1, Gfapα, Bdnf, and Tnfα was sensitive to variations in GbH, while memory impairment was not. Conclusions: This study highlights the importance of considering environmental factors in studies using transgenic animal models. The obtained data suggests that GbH can influence the parameters of interest in preclinical research, implicating the need for the optimization of future study designs.

Tissue-specific consequences of tag fusions on protein expression in transgenic mice

2025-06-18

Gillian C.A. Taylor , Lewis Macdonald , Natalia A. Szulc +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract Genetic fusion of protein tags is widely used to study protein functions in vivo . It is well known that tag fusion can cause unwanted changes in protein stability, … Abstract Genetic fusion of protein tags is widely used to study protein functions in vivo . It is well known that tag fusion can cause unwanted changes in protein stability, but whether this is an inherent property of the tagged protein, or can be influenced by the cell and tissue environment, is unclear. Using a series of genome edited mouse models, we show that tag-dependent changes in protein expression can vary across different primary cell and tissue contexts. In one case ( Ncaph2 ), a C-terminal auxin-inducible degron fusion strongly increased protein stability in some tissues but decreased it in others. Destabilisation resulted from tissue-specific ‘leakage’ of the auxin-inducible degron, which depended on TIR1 expression, and occurred selectively in the small intestine where basal concentrations of auxin / indole-3-acetic acid can reach levels that are sufficient to trigger protein degradation in cultured cells. Stabilisation occurred in post-mitotic cells via an endogenous degradation signal situated at the Ncaph2 C-terminus, which normally undergoes activation upon cell cycle exit, but is inactivated by C-terminal tag fusion. Our results highlight the underappreciated importance of cell and tissue environment in determining the consequences of tag fusions on protein expression, which may be particularly important in animal models that contain diverse cell types.

Establishment of Dental Pulp Cell Culture System for Analyzing Dentinogenesis in Mouse Incisors

2025-06-18

Yuka Kato , Insoon Chang , Satoshi Yokose | Dentistry Journal

Background: The dentin-pulp complex plays a vital role in tooth health. Dentin forms the main body the tooth and continues to form throughout life to maintain homeostasis and provide protection … Background: The dentin-pulp complex plays a vital role in tooth health. Dentin forms the main body the tooth and continues to form throughout life to maintain homeostasis and provide protection against deleterious external stimuli. However, the detailed mechanism of dentin formation remains poorly understood, and there is a need for new regenerative therapies. This study therefore investigated whether primary dental pulp cells from mice could be used to establish a new culture system. Methods: Mouse mandibles were divided along the ramus to extract dental pulp tissue containing cervical loops. The extracted tissue was cultured in an incubator to promote cell out-growth and increase the number of cells available for experimentation. Results: Cultured cells formed mineralized nodules, confirmed by Alizarin red S staining. The expression levels of dentin sialo protein, bone gamma-carboxyglutamate protein, and type I collagen mRNAs in cultured dental pulp cells on day 15 were lower than those in intact mouse dental pulp tissue, and the expression of all mRNAs was confirmed through electrophoresis. Conclusions: This study established a primary culture system using dental pulp tissue extracted from mouse mandibular incisors. The results demonstrated that dental pulp cells can differentiate into odontoblast-like cells and form dentin-like mineralized nodules, thereby offering a useful system for studying dentin formation and odontoblast differentiation.

Nutritional Growth Enhancement of G4 Transgenic Mutiara Catfish (Clarias gariepinus) Broodstock Candidate Using Mixed Feed

2025-06-17

Ibnu Dwi Buwono , Ine Maulina , Iskandar Iskandar +1 more | Asian Journal of Biotechnology and Bioresource Technology

Transgenic mutiara catfish inserted with an additional gene (CgGH) exhibit faster growth compared to non-transgenic mutiara catfish. This potential for rapid growth also needs to be supported by proper nutrition. … Transgenic mutiara catfish inserted with an additional gene (CgGH) exhibit faster growth compared to non-transgenic mutiara catfish. This potential for rapid growth also needs to be supported by proper nutrition. This study aimed to determine the optimal feed dosage using a mixture of HI-PROVITE 781 pellets and boiled tuna (Euthynnus affinis) flakes to evaluate growth efficiency and feed cost efficiency in sub-adult size G4 transgenic Mutiara catfish (5 months old) as they approach broodstock size. The research used a Completely Randomized Design (CRD) with 4 treatments and 3 replications. The treatments consisted of different doses of HI-PROVITE 781: treatment A (2.5%), B (2%), C (1.5%), and D (2.5% as control: non-transgenic mutiara catfish), based on the biomass weight of the test fish. Additionally, 10 g of boiled tuna flakes were added to each treatment for 42 days. The observed parameters included average weight gain (AWG), feed convertion ratio (FCR), and feed cost. The results showed that the best feed mixture of HI-PROVITE 781 pellets and boiled tuna flakes was found in treatment B (2% dose + 10 g), with an average weight gain of 1,515.00 g ± 422.26 g (3.35 times increase); FCR of 1.14 ± 0.11; feed cost of IDR 17,944.32 and the highest net income from feed at IDR 76,905.68.

Development of an allogenic in-ovo assay for primary chicken follicle stimulation

2025-06-16

Sabine Klein , Wilfried A. Kues | Theriogenology

Here, we established an optimized co-culture for primordial and primary chicken follicles implanted into the chorioallantoic membrane of cultured eggs with growing embryos in surrogate shells (CAM-assay) for direct access … Here, we established an optimized co-culture for primordial and primary chicken follicles implanted into the chorioallantoic membrane of cultured eggs with growing embryos in surrogate shells (CAM-assay) for direct access during their initial growth and germline specification. The germline specification is initiated by germ plasm accumulation in the Balbiani body of early primordial follicles directed by the maternally provided germ plasm organizer Bucky ball. To investigate the regulatory network during this initial germline specification, we developed a protocol for the in-vitro lipofection of separated primordial and primary follicles with interfering RNAs. The implantation of lipofected follicles in the nourishing environment of the CAM of preincubated eggs and in-ovo culture of these stimulated follicles for further 2-4 days could be used to investigate alterations in the resulting transcription and protein profiles. To ensure optimized developmental conditions for the implanted follicles, either preincubated eggs fenestrated with a shell opening of about one square centimeter or preincubated eggs transferred in surrogate shells through an opening of 35 mm diameter, were compared as recipients. The development of the follicles in a three-dimensional culture system using surrogate shells ensures maximum access to the surface of the CAM and an exceptionally high vitality of the recipient embryos as well as the implanted follicles. Phenotypical parameters for the vitality assessment of the follicles after 2-4 days of co-incubation are provided. First data of interference effects with siRNAs for Bucky ball and CVH are added.

Humanized mice to model rare human diseases

2025-06-16

Larry J. Suva | Proceedings of the National Academy of Sciences

Revisão: Lipossomas de lecitina de soja na criopreservação do sêmen canino

2025-06-16

Edenara Anastácio da Silva , Carine Dahl Corcini , Izani Acosta Bonel +1 more | OBSERVATÓRIO DE LA ECONOMÍA LATINOAMERICANA

A criopreservação visa manter a funcionalidade e viabilidade espermática para aplicação em biotécnicas reprodutivas, porém o processo expõe os espermatozoides à crioinjúrias, danos estruturais e funcionais devido à redução da … A criopreservação visa manter a funcionalidade e viabilidade espermática para aplicação em biotécnicas reprodutivas, porém o processo expõe os espermatozoides à crioinjúrias, danos estruturais e funcionais devido à redução da temperatura, além de estresse químico e oxidativo. O objetivo deste estudo é descrever os principais trabalhos já publicados que relacionam o uso de lipossomas de lecitina de soja na criopreservação de sêmen canino, bem como discorrer sobre os principais aspectos e aplicabilidade do assunto. A utilização de lipossomas de lecitina de soja em diluentes de criopreservação seminal em cães se demonstra promissora surge como uma alternativa ao uso de aditivos de origem animal, como a lipoproteína de baixa densidade (LDL) proveniente da gema de ovo, tendo como principal efeito citado a proteção da membrana plasmática espermática. Porém são necessários estudos mais aprofundados a fim de, determinar a concentração ideal das diferentes lecitinas, padronização do método de adição aos diluentes, bem como análises mais aprofundadas de viabilidade e funcionalidade espermática in vivo.

Assessment of the Genotoxic Hazard of Estuarine Sediments Using an Integrative Approach With LacZ Plasmid-Based Transgenic Mice.

2025-06-16

Miguel Pinto , Joana Sacadura , Pedro M. Costa +3 more | PubMed

Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the … Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the genotoxic potential of a contaminated sediment sample from an urban and industrial area of the Sado Estuary, by combining the analysis of multiple endpoints in the LacZ plasmid-based transgenic mouse model exposed for 28 days to contaminated estuarine sediment extracts through drinking water. The DNA and chromosome damaging effects were monitored in peripheral blood at 7-day intervals using the standard and enzyme-modified Comet assay, as well as the micronucleus assays in peripheral blood cells. After euthanasia, DNA damage was analyzed in several mouse tissues, and LacZ mutant frequencies were determined in the liver. Livers were also surveyed for histopathological analysis. A time-dependent increase in micronuclei frequency was seen at all tested doses, in spite of no induction of DNA damage in any organ or mutation induction in the liver of exposed mice. The liver from mice exposed to sediment extracts did not reveal major alterations besides evidence of inflammation. Overall, the integration of the endpoints analyzed in the mice is suggestive of potential chronic, rather than acute, adverse effects in vivo, and points to the need for further research in the resident human population in the area. This experimental design can be used to assess the genotoxicity of complex environmental mixtures, understand how they work, and reduce costs and resources while speeding up data collection and interpretation.

Single-cell omics reveal distinct gene regulatory dynamics underpinning embryonic and extraembryonic lineage functions during pig blastocyst development

2025-06-15

Adrien Dufour , Marie‐Noëlle Rossignol , Patrick Manceau +7 more | bioRxiv (Cold Spring Harbor Laboratory)

The late-stage development of the blastocyst before implantation is a unique feature of ungulates. During this period, the epiblast proliferates and remains pluripotent for several days before gastrulation begins. Simultaneously, … The late-stage development of the blastocyst before implantation is a unique feature of ungulates. During this period, the epiblast proliferates and remains pluripotent for several days before gastrulation begins. Simultaneously, extra-embryonic tissues undergo significant growth, elongating to several tens of centimeters. However, the mechanisms that regulate and synchronize these processes remain poorly understood. In this study, we analyzed transcriptomic and epigenomic data at the single-cell level from early and late porcine blastocyst cells, spanning from the hatched blastocyst stage (E7) to early (E9) and late ovoid blastocyst (E11). We characterized 15,370 blastocyst cells, clustering them into distinct embryonic and extra-embryonic populations with specific chromatin accessibility landscapes. For each population, we inferred gene regulatory networks based on enhancer-driven gene regulation modules (eRegulons) and validated them through motif occupancy visualization. Our findings reveal strong dynamics in gene regulatory module activity within extra-embryonic tissues at the onset of blastocyst elongation, while gene regulatory activity in the epiblast remains relatively stable.

Morula complementation restores male germline in NANOS2 null sheep

2025-06-14

Zachariah L. McLean , Lisanne M. Fermin , Sarah Jane Appleby +7 more | PNAS Nexus

Abstract Current livestock breeding is slow to respond to rapidly mounting environmental pressures that threaten sustainable animal protein production. New approaches can accelerate genetic improvement by multiplying valuable embryonic, rather … Abstract Current livestock breeding is slow to respond to rapidly mounting environmental pressures that threaten sustainable animal protein production. New approaches can accelerate genetic improvement by multiplying valuable embryonic, rather than adult genotypes. Chimaeras, derived from complementing a sterile host with a fertile donor embryo, provide a pathway to multiply and exclusively transmit elite male germlines. We established genetically sterile hosts and optimized embryo complementation conditions to achieve absolute germline transmission in sheep. The spermatogonial-specific gene NANOS2 was disrupted in male (NANOS2+/-, NANOS2-/-) and female (NANOS2-/-) ovine fetal fibroblasts via gRNA-Cas9-mediated homology-directed repair. Targeted cell strains and wild-type controls were used to produce cloned offspring for breeding and phenotyping. Male homozygous knockout clones lacked detectable germ cells, while the somatic compartment of the testis remained intact. By contrast, male monoallelic and female biallelic targeting of NANOS2 did not affect germline development, resulting in fertile animals capable of producing fertile offspring with normal reproductive performance. The germ cell niche in NANOS2-/- hosts was most efficiently complemented by aggregating compacted morulae, rather than earlier cleavage stages, resulting in 97% blastocyst chimaerization. Embryo-complemented cloned lambs from two different donor cell lines showed variable chimaerism across tissues from each germ layer, including various degrees of germline colonization. A subset of germline chimaeras contained normal numbers of prospermatogonia, indicating that the germline was fully restored for absolute transmission of the donor cell genotype.

1851-P: AAV Capsid Variant Screen for Building a Scalable Methodology to Modify Human Islet Gene Expression

2025-06-13

SAYRO JAWUREK , ALEXANDRA C. TITLE , FELIX FORSCHLER +3 more | Diabetes

Introduction and Objective: Loss of functional β-cell mass in pancreatic islets is a key factor in diabetes etiology. Isolated human islets, the gold standard for islet research, pose experimental challenges … Introduction and Objective: Loss of functional β-cell mass in pancreatic islets is a key factor in diabetes etiology. Isolated human islets, the gold standard for islet research, pose experimental challenges due to their variable size, composition, function and purity, as well as their low viability and functionality in prolonged culture. Additionally, their genetic manipulation is impeded by poor penetration of transduction reagents, including viral particles. Methods: To address these challenges, we developed a scalable adeno-associated virus (AAV) mediated transduction methodology using reaggregated human islet microtissues (MTs), enabling high transduction efficiencies and organotypic islet architecture and function for over 28 days in culture. We tested nine AAV capsids to identify the variant with the best affinity to transduce islets, with minimal effect on islet MT function and viability. DYRK1A, a previously characterized target known to induce islet cell proliferation, was then knocked down, while harmine, a potent DYRK1A inhibitor, served as positive control. Islet MTs were stained in 3D for DAPI, NKX6.1 and EdU, imaged on a high-content confocal microscope, and analyzed for total and β-cell counts and fraction of proliferating β- and non-β-cells using an automated 3D analysis pipeline. Results: AAV2.7m8 and KP1 exhibited the highest transduction efficiencies and transgene expression with minimal impact on insulin secretion, content, and ATP levels. Despite a general decrease in cellular proliferation following viral transduction, DYRK1A knockdown via AAV2.7m8 increased total proliferation within human islet MTs but was unable to induce proliferation in β-cells, while harmine was effective in inducing proliferation in both β-and non-β-cells. Conclusion: In summary, we developed a scalable and high efficiency platform for AAV-mediated gene expression modification in human islets, while maintaining stable islet function. This platform enables the study of putative therapeutic targets directly in human islets. Disclosure S. Jawurek: None. A.C. Title: None. F. Forschler: Employee; InSphero AG. C. Rufer: None. T. Klein: Employee; Boehringer-Ingelheim. B. Yesildag: Other Relationship; Novo Nordisk, Boehringer-Ingelheim, Eli Lilly and Company, Biomea Fusion, Biosplice Therapeutics, Abata Therapeutics, AstraZeneca, Amgen Inc.

Investigation of A2 allele frequency in Taiwanese Holstein cattle using genetictes ting

2025-06-13

J.W. Shiau , C. H. Chao | Polish Journal of Veterinary Sciences

β-Casein accounts for approximately 30% of total milk protein, with the A1 and A2 variants being the most common. A1 β-casein may release β-casomorphin-7 (BCM-7) during digestion, which is associated … β-Casein accounts for approximately 30% of total milk protein, with the A1 and A2 variants being the most common. A1 β-casein may release β-casomorphin-7 (BCM-7) during digestion, which is associated with adverse health effects, whereas A2 β-casein does not. This study investigated the A2 allele frequency and its potential influence on milk performance in Holstein cattle in Taiwan. A total of 1,050 cows from five herds were genotyped using the GeneSeek 50K SNP chip. The A2 allele frequency ranged from 0.58 to 0.75 among herds, with an average of 0.66. Hardy-Weinberg equilibrium tests indicated no significant deviation within herds. Pedigree validation confirmed Mendelian inheritance of A1 and A2 alleles. Furthermore, 348 cows with complete lactation records were evaluated for 305-day mature equivalent milk and fat yields across genotypes. Although cows with the A1/A1 genotype showed numerically higher milk and fat yields, one-way ANOVA and Tukey's HSD tests revealed no statistically significant differences. These results suggest that while A2 allele selection is increasing in Taiwan, β-casein genotype does not significantly influence milk or fat production in the studied population.

Fin Cells as a Promising Seed Cell Source for Sustainable Fish Meat Cultivation

2025-06-12

Zhihua Du , Jihui Lao , Yuyan Jiang +7 more | Foods

Cell-cultured meat production relies on stable, proliferative seed cells, commonly sourced from muscle satellite cells (MuSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). However, establishing such cell lines in fish species … Cell-cultured meat production relies on stable, proliferative seed cells, commonly sourced from muscle satellite cells (MuSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). However, establishing such cell lines in fish species remains technically challenging. While pluripotent stem cells (e.g., ESCs/MSCs) offer alternatives, their differentiation efficiency and predictability are limited. Here, we developed TCCF2022, a novel caudal fin-derived cell line from Topmouth culter (Culter alburnus), which expresses pluripotency markers (AP, Oct4, Sox2, Klf4, and Nanog) and aggregated growth to form 3D spheroids. Forskolin supplementation enhanced pluripotency maintenance. The presence of adipogenic and myogenic lineage cells within the 3D spheroids was confirmed, demonstrating their potential as seed cells for cell-cultured meat. Using a small-molecule cocktail 5LRCF (5-Azacytidine, LY411575, RepSox, CHIR99021, and Forskolin), we successfully differentiated TCCF2022 cells into functional myotubes. Additionally, we established a method to induce the differentiation of TCCF2022 cells into adipocytes simultaneously. Thus, the TCCF2022 cell line can be used to improve muscle fiber formation and lipid composition, potentially enhancing the nutritional profile and flavor of cultured fish meat.

Extended and cryopreserved semen of Banaba native chicken with sugarcane extract for artificial insemination

2025-06-11

Salifu A-R. S. , Geleo Dichoso , Percival Sangel | Deleted Journal

The experiment was conducted to test the efficacy of sugarcane extract extender (40% SE+30% dH2O (distilled water) + 30% EY-C (Egg-yolk citrate)) developed in our laboratory and semen cryopreserved with … The experiment was conducted to test the efficacy of sugarcane extract extender (40% SE+30% dH2O (distilled water) + 30% EY-C (Egg-yolk citrate)) developed in our laboratory and semen cryopreserved with SE extender with 3% glycerol (SEG) as a cost-effective alternative in an in vivo fertility trial using a practical farm approach. The lactated ringers’ solution (LRS) served as a control. The cryopreserved semen was thawed at room temperature (25-27°C) for 15-20 min for the artificial insemination. Thirty-six (36) 7.5 month old Dekalb White hens with an average weight of 1.65 kg were divided into three groups of 12 hens per group and housed individually in a completely randomized design (CRD). The groups: 1, 2 and 3 were artificially inseminated with LRS extended semen, SE extended semen and SEG cryopreserved semen respectively. A total of 284 eggs were collected and incubated, consisting of 95 (group 1), 89 (group 2) and 100 (group 3). The results showed that, fertility rates of LRS extended semen (60.04 ± 1.83%) and SE extended semen (56.94±2.62%) were significantly (p≤0.05) higher than the cryopreserved semen (25.56±3.22%). However, hatchability rates were statistically similar at 5% among the treatment means for the LRS (51.26±4.15%), SE (62.02±6.34%) and SEG (49.31±2.86%). The findings of this experiment suggest that sugarcane extract with 3% glycerol successfully produced fertile eggs and viable chicks. The extender showed no adverse effects on fertility or hatchability, indicating its potential as a promising medium for avian semen cryopreservation. The SE and SEG can be used for poultry propagation, breeding and conservation.