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Life
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Gene Editing of Large Mammals
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Gene Editing of Large Mammals

A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Animal Science".

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 19993

Special Issue Editors

Dr. Zhengxing Lian

E-Mail Website
Guest Editor
Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Interests: gene editing; sheep; reproduction; innate immunity
Dr. Shou-Long Deng

E-Mail Website
Guest Editor
Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100006, China
Interests: SSCs differentiation; mammalian oocytes; somatic cell clone; innate immunity
Special Issues, Collections and Topics in MDPI journals
Dr. Xuguang Du

E-Mail Website
Guest Editor
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
Interests: pig; human disease model; xenotransplantation

Special Issue Information

Dear Colleagues,

Genome editing technologies can be used to precisely alter genomic sequences and modify the genetic information at specific target sites of an organism. Compared with the long breeding cycle and high cost of traditional genetic breeding, gene editing can be used to implement cost-effective targeted editing of animal genomes, thereby providing a powerful application tool for rapid improvement of the production traits of large mammals. In addition, some large mammals are closely related to human beings. These gene-edited large mammals can serve as a disease model, animal bioreactor, or be used in xenotransplantation. However, the application of gene editing to generate gene-edited large mammals is still in a stage of significant development. To stimulate progress on this topic, this Special Issue aims to cover the following non-exhaustive areas of relevance:

The application of gene editing to large mammals and the successful generation of mutants;

How to improve gene editing efficiency in large animals;

Developing disease models using gene-editing technology;

Application of novel gene-editing techniques in xenotransplantation, animal bioreactor and humanized organ research.

Dr. Zhengxing Lian
Dr. Shoulong Deng
Dr. Xuguang Du
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Life is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • gene editing
  • large mammals
  • molecular breeding
  • application prospect

Published Papers (6 papers)

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Research

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16 pages, 1761 KiB  
Article
Progesterone Promotes In Vitro Maturation of Domestic Dog Oocytes Leading to Successful Live Births
by Yumin Qin, Shenjiong Feng, Min Zheng, Xiaojuan Liu, Jianping Zhao, Qintao Zhao, Junhua Ye, Jidong Mi and Yougang Zhong
Life 2022, 12(11), 1778; https://doi.org/10.3390/life12111778 - 3 Nov 2022
Cited by 3 | Viewed by 2233
Abstract
Gene-edited dogs are promising models for biomedical research because they have hundreds of genetic diseases that are similar to humans. A common method for producing gene-edited dogs is assisted reproductive technology (ART) using in vivo oocytes or embryos, but it is much more [...] Read more.
Gene-edited dogs are promising models for biomedical research because they have hundreds of genetic diseases that are similar to humans. A common method for producing gene-edited dogs is assisted reproductive technology (ART) using in vivo oocytes or embryos, but it is much more inefficient and has a higher cost. ART for dogs has lagged mostly because of the lack of an efficient in vitro maturation system. Because early maturation of canine oocytes occurs in follicles with extremely high concentrations of progesterone (P4), we hypothesize that P4 has an important role during maturation. In this study, we obtained ovaries of female dogs and collected cumulus–oocyte complexes, which were cultured in vitro in microdrops containing different P4 concentrations (0, 10, 40, 100 or 200 µg/mL). We found that 40 µg/mL P4 produced the highest oocyte maturation rate (29.7% ± 7.1%, p < 0.05). We also evaluated the quality of in vitro matured oocytes by in vitro fertilization and single-cell RNA sequencing, and both indicated an improvement in oocyte developmental potential. In conclusion, we successfully obtained the first live dogs using in vitro matured oocytes by adding P4 to optimize the in vitro maturation system of canine oocytes, and established a new and low-cost method to produce dogs via in vitro maturation and in vitro fertilization. Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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18 pages, 10275 KiB  
Article
Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage
by Jing Wang, Sami Ullah Khan, Pan Cao, Xi Chen, Fengchong Wang, Di Zou, Honghui Li, Heng Zhao, Kaixiang Xu, Deling Jiao, Chang Yang, Feiyan Zhu, Yaxuan Zhang, Yanhua Su, Wenmin Cheng, Baoyu Jia, Yubo Qing, Muhammad Ameen Jamal, Hong-Ye Zhao and Hong-Jiang Wei
Life 2022, 12(5), 630; https://doi.org/10.3390/life12050630 - 24 Apr 2022
Cited by 1 | Viewed by 2682
Abstract
As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the [...] Read more.
As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from Diannan miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-β1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases. Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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21 pages, 2645 KiB  
Article
Growth Traits and Sperm Proteomics Analyses of Myostatin Gene-Edited Chinese Yellow Cattle
by Yuefang Zhao, Lei Yang, Guanghua Su, Zhuying Wei, Xuefei Liu, Lishuang Song, Chao Hai, Di Wu, Zhenting Hao, Yunxi Wu, Li Zhang, Chunling Bai and Guangpeng Li
Life 2022, 12(5), 627; https://doi.org/10.3390/life12050627 - 23 Apr 2022
Cited by 10 | Viewed by 3824
Abstract
Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. [...] Read more.
Chinese Yellow Cattle, an ancient and domesticated breed for draft service, provide unique animal genetic resources with excellent genetic features, including crude feed tolerance, good stress resistance, strong adaptability, and tender meat quality; however, their production performance and meat yield are significantly inferior. Herein, the myostatin gene (MSTN), a negative regulator of skeletal muscle development, was knocked out by CRISPR/Cas9 technology. Eight MSTN gene-edited bull calves (MT) were born, and six of them are well-developed. Compared with the control cattle (WT), the growth trait indexes of MT cattle were generally increased, and the hindquarters especially were significantly improved. The biochemical indexes and the semen characteristics demonstrated that MT bulls were healthy and fertile. Consistent with our conjecture, the wobble and beating of MT bull spermatozoa were significantly higher than that of WT. Nine sperm motility-related proteins and nineteen mitochondrial-related proteins were identified by up-regulation in MT bull spermatozoa using FLQ proteomic technique and act to govern sperm flagellum assembly, organization, and beating and provide sufficient energy for sperm motility. The current study confirmed that the MSTN gene-edited Chinese Yellow cattle have improved growth traits and normal fertility, which can be used for beef cattle production and breeding. Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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15 pages, 1694 KiB  
Article
A 90-Day Safety Study of Meat from MSTN and FGF5 Double-Knockout Sheep in Wistar Rats
by Yue Zhao, Mingming Chen, Yao Li, Xueling Xu, Sujun Wu, Zhimei Liu, Shiyu Qi, Guang Yi, Xiaosheng Zhang, Jinlong Zhang, Xiaofei Guo, Kun Yu, Shoulong Deng, Yan Li and Zhengxing Lian
Life 2022, 12(2), 204; https://doi.org/10.3390/life12020204 - 29 Jan 2022
Cited by 4 | Viewed by 2554
Abstract
MSTN and FGF5 gene knockout sheep generated by the CRISPR/Cas9 system exhibit the ‘double-muscle’ phenotype, and increased density and length of hairs, providing valuable new breeding material. In a previous study, we obtained MSTN and FGF5 double-knockout sheep of significant breeding value. In [...] Read more.
MSTN and FGF5 gene knockout sheep generated by the CRISPR/Cas9 system exhibit the ‘double-muscle’ phenotype, and increased density and length of hairs, providing valuable new breeding material. In a previous study, we obtained MSTN and FGF5 double-knockout sheep of significant breeding value. In this study, we carried out a 90-day feeding study in Wistar rats to assess the safety of genome-edited mutton. Seven rat groups with 10 females and 10 males per group were fed different concentrations (3.75%, 7.5%, and 15%) of double-knockout mutton or wild-type mutton in a conventional commercial diet for 90 days. At the end of the feeding, routine urine and blood tests and measurements of blood biochemical indicators were performed. Furthermore, the major organs of each group of rats were weighed and examined histopathologically. Although there were significant differences among the groups in some parameters, all values were within the normal ranges. Therefore, the 90-day rat feeding study showed that the meat from MSTN and FGF5 double-knockout sheep did not have any long-term adverse effects on rat health. This study also provides valuable reference information for assessing the safety of meat from animals with knockout of multiple genes. Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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15 pages, 3086 KiB  
Article
Efficient Generation of P53 Biallelic Mutations in Diannan Miniature Pigs Using RNA-Guided Base Editing
by Honghui Li, Wenmin Cheng, Bowei Chen, Shaoxia Pu, Ninglin Fan, Xiaolin Zhang, Deling Jiao, Dejia Shi, Jianxiong Guo, Zhuo Li, Yubo Qing, Baoyu Jia, Hong-Ye Zhao and Hong-Jiang Wei
Life 2021, 11(12), 1417; https://doi.org/10.3390/life11121417 - 17 Dec 2021
Cited by 3 | Viewed by 2920
Abstract
The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than [...] Read more.
The base editing 3 (BE3) system, a single-base gene editing technology developed using CRISPR/Cas9n, has a broad range of applications for human disease model construction and gene therapy, as it is highly efficient, accurate, and non-destructive. P53 mutations are present in more than 50% of human malignancies. Due to the similarities between humans and pigs at the molecular level, pig models carrying P53 mutations can be used to research the mechanism of tumorigenesis and improve tumor diagnosis and treatment. According to pathogenic mutations of the human P53 gene at W146* and Q100*, sgRNAs were designed to target exon 4 and exon 5 of the porcine P53 gene. The target editing efficiencies of the two sgRNAs were 61.9% and 50.0%, respectively. The editing efficiency of the BE3 system was highest (about 60%) when C (or G) was at the 5th base. Puromycin screening revealed that 75.0% (21/28) and 68.7% (22/32) of cell colonies contained a P53 mutation at sgRNA-Exon5 and sgRNA-Exon4, respectively. The reconstructed embryos from sgRNA-Exon5-5# were transferred into six recipient gilts, all of which aborted. The reconstructed embryos from sgRNA-Exon4-7# were transferred into 6 recipient gilts, 3 of which became pregnant, resulting in 14 live and 3 dead piglets. Sequencing analyses of the target site confirmed 1 P53 monoallelic mutation and 16 biallelic mutations. The qPCR analysis showed that the P53 mRNA expression level was significantly decreased in different tissues of the P53 mutant piglets (p < 0.05). Additionally, confocal microscopy and western blot analysis revealed an absence of P53 expression in the P53 mutant fibroblasts, livers, and lung tissues. In conclusion, a porcine cancer model with a P53 point mutation can be obtained via the BE3 system and somatic cell nuclear transfer (SCNT). Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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Review

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17 pages, 862 KiB  
Review
Application of Gene Editing Technology in Resistance Breeding of Livestock
by Sutian Wang, Zixiao Qu, Qiuyan Huang, Jianfeng Zhang, Sen Lin, Yecheng Yang, Fanming Meng, Jianhao Li and Kunli Zhang
Life 2022, 12(7), 1070; https://doi.org/10.3390/life12071070 - 18 Jul 2022
Cited by 9 | Viewed by 4052
Abstract
As a new genetic engineering technology, gene editing can precisely modify the specific gene sequence of the organism’s genome. In the last 10 years, with the rapid development of gene editing technology, zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR/Cas9 systems have [...] Read more.
As a new genetic engineering technology, gene editing can precisely modify the specific gene sequence of the organism’s genome. In the last 10 years, with the rapid development of gene editing technology, zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR/Cas9 systems have been applied to modify endogenous genes in organisms accurately. Now, gene editing technology has been used in mice, zebrafish, pigs, cattle, goats, sheep, rabbits, monkeys, and other species. Breeding for disease-resistance in agricultural animals tends to be a difficult task for traditional breeding, but gene editing technology has made this easier. In this work, we overview the development and application of gene editing technology in the resistance breeding of livestock. Also, we further discuss the prospects and outlooks of gene editing technology in disease-resistance breeding. Full article
(This article belongs to the Special Issue Gene Editing of Large Mammals)
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