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Triple transgenic pigs suitable for xenograft

A technology for transgenic pigs, genes, applied in the field of xenotransplantation and genetic modification, which can solve problems such as expensive maintenance drugs, increased infection risk, weight gain, etc.

Pending Publication Date: 2017-08-29
INDIANA UNIV RES & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because immunosuppressive drug regimens suppress the patient's immune response, they increase the risk of infection, require expensive maintenance medications, can include drugs that interact with other drugs, and can cause additional side effects, such as weight gain

Method used

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  • Triple transgenic pigs suitable for xenograft
  • Triple transgenic pigs suitable for xenograft
  • Triple transgenic pigs suitable for xenograft

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1. DNA sequencing analysis of targeted CMAH, GGTA1 and β4GalNT2 regions

[0107] Genomic DNA from cloned pigs was extracted using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO). PCR amplification of CMAH, GGTA1 and β4GalNT2 Crispr / Cas9 target regions was performed. Primers were used to sequence the targeted CMAH, GGTA1 and β4GalNT2 regions.

[0108] Using Pwo Master (Roche, Indianapolis IN), Pwo SuperYield DNA polymerase, dNTPack (Roche Applied Science, Indianapolis, IN) was used. The PCR conditions for GGTA1 were as follows: 94°C, 2 minutes; 94°C, 15 seconds, 54°C, 30 seconds, and 72°C, 45 seconds, for 15 cycles; 94°C, 15 seconds, 54°C, 30 seconds, 72°C , 45 seconds, plus 5 seconds per cycle, for 25 cycles; and a final extension step of 72° C. for 5 minutes. For CMAH, 94°C for 2 minutes; 94°C for 15 seconds, 56°C for 30 seconds and 72°C for 45 seconds for 15 cycles; 94°C for 15 seconds, 56°C for 30 seconds, 72°C for 45 secon...

Embodiment 2

[0110] Example 2. Generation of knockout pigs (triple transgenic pigs)

[0111] Oligonucleotide annealing was performed using Addgene plasmid 42230 [http: / / www.addgene.org / 42230 and 20] and cloned into the PX330 plasmid to drive gRNA expression. The oligonucleotide pair targeting the gene is GGTA1 (NCB1 accession number: XM_005660398.1), 5'CACCGAGAGAAAATAATGAATGTCAA-3' forward) (SEQ ID NO: 8), 5'AAATTGACATTCATTATTTTCTC-3' (reverse) (SEQ ID NO: 9); CMAH (NCBI accession number: NM_001113015.1) 5'-CACCGAG AAGGTACGTGATCTGT-3' (forward) (SEQ ID NO: 10), 5'-AAACACAGATCACGTACCTTACTC-3' (reverse) (SEQ ID NO: 11; β4GalNT2 (NCBI accession number NM_001244330.1) 5'-CACCGTGTATCGAGGAACACGCTT-3' (forward) (SEQ ID NO: 12), 5'-AAACAAGCGTGTTCCTCGATACAC-3' (reverse) (SEQ ID NO: 13 ).

[0112] Hepatic-derived cells were co-transfected with all three gRNA / Cas9 plasmids. After 48 hours, the treated cells were passed through an IB4 lectin column to isolate α-Gal null cells. Two million α-Gal ...

Embodiment 3

[0115] Example 3. IB4 counter-selection for triple knockout

[0116] Liver-derived cells (LDCs) were transfected with three sets of targeting constructs (αGal, β4GalNT2 and CMAH). Cells were selected with IB4, a substance that binds αGal. Cellular DNA from a large population of cells surviving IB4 counter-selection was obtained and evaluated for target gene sequences. A large population of cells surviving the IB4 counter-selection was used directly for SCNT to make pregnant pigs.

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Abstract

The application provides methods of improving a rejection related symptom, reducing premature separation and methods of producing a compound of interest with an altered epitope profile are provided. Knockout pigs with a disrupted gene or genes, and porcine organs, tissues, and cells therefrom are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No: 62 / 067,129, filed October 22, 2014, the contents of which are incorporated herein by reference. [0003] Incorporation of Sequence Listings [0004] The sequence listing in text format filed with this document is hereby incorporated by reference in its entirety for all purposes. [0005] Statement Regarding Federally Sponsored Research or Development [0006] Not applicable. technical field [0007] The present invention generally relates to methods for developing transgenic pigs, transgenic pig organs, tissues or cells suitable for transplantation into humans, especially those with reduced propensity to cause thrombocytopenia, hyperacute rejection (HAR) or platelet uptake transgenic pigs) in the field of xenotransplantation and genetic modification. Background technique [0008] It is well known that transplantation from one animal to another of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/12A01K67/027
CPCA61K35/12A01K2217/075A01K2217/15A01K2227/108A01K2267/025A61K35/407C12N2517/02A01K67/0276A61P17/02A61P43/00A61K35/22
Inventor 约瑟夫·A·泰克托尔
Owner INDIANA UNIV RES & TECH CORP
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