Pigs have been considered as the ideal models for human diseases and xenotransplantation, because of their close similarity to humans in terms of organ size, anatomy, physiology, metabolism and immunology. The genome needs to be modified to achieve pig models with a specific application. Technologically, direct genetic modification in large animals like pigs has not been applicable. Currently, generation of gene-edited pigs mostly relies on either fertilized egg injection or somatic cell nuclear transfer approaches. For fertilized egg injection, many of the resulting founder animals were chimeric ones with multiple mutations. One or two more rounds of further breeding and two to three more years are needed to acquire animals with a practical application. For somatic nuclear transfer approach, the efficiency has been very low. It is also very expensive and time consuming.

In this study, using gene-editing technology, researchers inserted a genome-cutting protein (Cas9) into porcine Rosa26 loctus, meaning that a gene scissor was permanently mounted into pig genomes. Cas9 gene was flanked with lox Psites, which allowed expression of Cas9 to be controlled by Cre recombinase, playing a role of a switch turning on or off the gene scissors. With this model, the scientists can directly edit porcine genomes by introducing gRNA and Cre recombinase into the pigs, and facilitate the process of creating genome edited pigs.

The lentivirus containing Cre recombinase and sgRNAs targeting six tumor associated genes were introduced to the lungs of these pigs by intranasal delivery method. Three months after the infection, the Cre-dependent Cas9-expressing pigs presented lung cancer symptoms and pathological lesions, demonstrating that large animal models with primary lung tumors were successfully generated.

This Cre-dependent Cas9-expressing pig could provide a simple and efficient way to produce a variety of in vivo genetic modifications, including single-gene knockout, multi-gene knockout, chromosome rearrangement, and large fragment deletion, which should substantially promote studying gene functions, and facilitate the establishment of genetically modified pigs with valuable applications in both biomedicine and agriculture.

On Nov 16, Dr. Lai Liangxue’s group at the Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, published an article entitled “Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing” online in Genome Research. In this work, they generated Cre-dependent Cas9-expressing pigs as a versatile large animal model. With this model, they accomplished direct in vivo gene editing in pigs, and for the first time, established a large animal primary lung cancer model.

For more information, please contact:

Dr. Lai Liangxue

E-mail:  lai_liangxue@gibh.ac.cn

Guangzhou Institute of Biomedicine and Health

Chinese Academy of Sciences

Source: Guangzhou Institute of Biomedicine and Health, CAS