Identification and cloning of the porcine ROSA26 promoter and its role in transgenesis

Li, Ping; Burlak, Christopher; Estrada, José L.; Cowan, Peter J.; Tector, A. Joseph

doi:10.7243/2053-6623-2-1

Cited by 3 publications

(2 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The sequence for synthesis of HDR and ROSA26 was first confirmed by Sanger sequencing. Previously published sequencing for the promoter region and exon 1 of the porcine ROSA26 locus was used to complete the gene template (54). The target sequence was interrogated against the porcine genome in the NCBI database using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and a sequence similar to the NC_010455.4 (72802131 to 72802975) located at chromosome 13 in the Sus scrofa genome (S. scrofa).…”

Section: Creation Of Hla-g1 Template and Grnas Specific To Ggta1 And Rosa26mentioning

confidence: 99%

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

et al. 2021

Self Cite

View full text Add to dashboard Cite

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

show abstract

Section: Creation Of Hla-g1 Template and Grnas Specific To Ggta1 And Rosa26mentioning

confidence: 99%

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The porcine ROSA26 locus has previously been targeted (Li S. et al, 2014), revealing ubiquitous expression of inserted transgenes in living pigs and that the strength of the porcine ROSA26 promoter is comparable to the H-2K b promoter (Li P. et al, 2014).…”

Section: Rosa26 Targeting and Expression Of Ho1mentioning

confidence: 99%

Multi‐transgenic pigs for xenotransplantation

Fischer

Kraner-Scheiber

Flisikowski

et al. 2013

Xenotransplantation

View full text Add to dashboard Cite

Genetically modified pigs offer a solution to the severe shortage of organs available for human transplantation. Inactivation of the α1,3‐galactosyltransferase gene, responsible for α1,3‐Gal epitope synthesis (1–2) was a major breakthrough and essentially solved the problem of hyperacute rejection. However, further modifications of donor pigs are required to enable long term xenograft survival. These include expression of complement regulatory, antithrombotic, endothelium protective and immuno‐regulatory transgenes. Current evidence indicates that complement regulator transgenes, such as CD46 and CD55, must be expressed at least fivefold more than their human endogenous equivalent to effectively protect grafted tissue (3). To explore means of achieving high expression, we produced a series of BAC‐ and PAC‐vector constructs containing different sized genomic complement regulatory genes, from 70 to 190 kb, under the control of endogenous or CAGGs (CMV enhancer chicken β actin) promoter sequences. Using these vectors we obtained high levels of CD46 and CD55 expression in vitro. To prepare multi‐transgene “packages” for introduction into animals, the most effective constructs were combined with one or two further xenoprotective transgenes, including A20 (4), HO‐1 (5), thrombomodulin (6) and CTLA4‐Ig (LEA 29Y). In cotransfection experiments single cell clones expressing up to five different xenogenes could be detected. Achieving desired levels of transgene expression in an animal is not only a function of the transgene construct, but also of the location at which it integrates. Position effect variation in expression of random transgenes is well‐documented. Transgene placement by gene targeting at a permissive locus offers a useful means of achieving reliable transgene expression. In mice the ROSA26 locus is widely used to support ubiquitous expression of inserted transgenes (7–8). Murine ROSA26 encodes no functional genes and extensive gene targeting of this locus has not led to deleterious effects on development or physiology (9). To enable a similar approach in pigs, we recently identified a strong candidate for the porcine ROSA26 locus. Gene targeted placement of a neomycin reporter gene construct under the control of the ROSA26 promoter showed expression in all examined porcine tissues of all three germ layers. This strongly suggests that porcine ROSA 26 may resemble the murine locus as a suitably permissive site for transgene expression. We are currently proceeding with gene targeting to place various xenoprotective transgene constructs at this locus. References: 1. Dai Y, Vaught TD, Boone J et al. Targeted disruption of the alpha1,3‐ galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 2. Lai L, Kolber‐Simonds D, Park K et al. Production of alpha‐1,3‐galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 3. Miyagawa S, Fukuta D, Kitano E et al. Effect of tandem forms of DAF(CD55) on complement‐mediated xenogeneic cell lysis. Xenotransplantation 20...

show abstract

Microstructure and Fracture Toughness of an Aluminum-Steel Impact Weld and Effect of Thermal Exposure

Kohlhorst

Kapil

Chen

et al. 2021

Metall Mater Trans A

View full text Add to dashboard Cite

Welding of aluminum alloys to steel is increasingly important in manufacturing, however the use of fusion welding is difficult because of disparate melting points and the possibility of intermetallic compound (IMC) formation. Here, an impact welding technique, vaporizing foil actuator welding, was utilized to produce solid-state joints between AA1100-O and 1018 mild steel. The relationship between weld processing conditions, microstructure, and mechanical properties were investigated. For this purpose, the welds were annealed between 300°C and 600°C and a combination of optical and scanning electron microscopy, along with image analysis was performed to characterize the weld microstructure and monitor IMC growth. Wedge testing was applied to understand the effect of annealing on the weld fracture toughness. A numerical model incorporating the Fick's laws of diffusion, grain boundary diffusion, and grain growth kinetics was also developed to simulate the IMC growth. The heterogeneity in the original microstructure caused persistent differences in IMC growth, as initial IMC seemed to increase nucleation and growth. Simulation results indicated short circuit diffusion to be the major contributor to IMC growth since it is consistently faster then experimental IMC growths compared with the computational results that used lattice diffusion only. Wedge testing reveals increased weld toughness for modest anneals of 300°C, possibly due to homogeneity at the weld interface while avoiding IMC growth.

show abstract

Identification and cloning of the porcine ROSA26 promoter and its role in transgenesis

Cited by 3 publications

References 25 publications

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses

Multi‐transgenic pigs for xenotransplantation

Microstructure and Fracture Toughness of an Aluminum-Steel Impact Weld and Effect of Thermal Exposure

Contact Info

Product

Resources

About