Cell and Tissue Journal

Abstract Introduction: Homeodomain transcription factor 1 (PDX-1) is necessary for proper pancreatic development and β-cell function. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, in conjunction with CRISPR-associated protein 9 (Cas9), facilitates modifications, particularly deletions, within the genome. The use of lentiviral vectors for gene transfer to somatic stem cells has increased significantly. They harbor advantageous features, including a relatively high gene loading capacity, a diminished likelihood of gene expression silencing following integration into the genome, and high efficiency in delivering genes to a variety of target cells. Furthermore, they elicit no undesirable immune reactions and offer a broad selection of alternative envelope proteins.
Aim: This study aims to introduce a mutation in the chicken PDX-1 gene. A guide RNA (gRNA) will be designed to complement a specific region of the PDX-1 gene. This approach aims to create targeted mutations with potential therapeutic applications in gene therapy.
 
Materials and Methods: The PDX-1 gene was cloned using the LentiCRISPRv2GFP vector. To delete and modify the PDX-1 gene, a specific single guide RNA (sgRNA) sequence is designed. This sgRNA sequence is approximately 100 base pairs (bp) in length, with about 76 bp derived from the scaffold segment already integrated into the LentiCRISPRv2GFP vector.
The region responsible for binding the sgRNA to the target genomic sequence is 20 nucleotides long and is complementary to 20 nucleotides in exon one of the PDX-1 gene. The CRISPR Design Tool (http://crispr.mit.edu) was used to design a specific sgRNA sequence targeting the PDX1 locus (sgPDX1).
The double-stranded oligonucleotide fragment and the LentiCRISPRv2GFP vector were subjected to enzymatic digestion using the BsmbI restriction enzyme. To create the LentiCRISPRv2GFP-sgPDX1 vector, the cleaved sgPDX1 sequence was joined with the cleaved vector. This was accomplished by mixing the vector and nucleotide fragment and subjecting them to a ligation reaction using the T4 ligase enzyme. The resulting vector contains a transcript of the sgRNA, specifically sgPDX1.
The heat shock method was employed to transform Escherichia coli Top10 host cells with the cloned LentiCRISPRv2GFP-sgPDX1 vector. To confirm the presence of the sgPDX-1 gene in the vector, a polymerase chain reaction (PCR) was conducted using primers targeted at a portion of the U6 promoter sequence (forward primer) and a segment downstream of the sgRNA gene region (reverse primer). Additionally, each PCR product was analyzed using enzymatic digestion with EcoRV and BsmBI enzymes. To confirm the successful cloning, the PCR product was sequenced.
Results: The PCR results showed an amplified fragment measuring 267 base pairs (bp), which indicated the presence of the sgPDX-1 gene. This was visualized on an agarose gel. Additionally, the reverse digestion with two restriction enzymes produced a 2000 bp fragment isolated from the vector. The sequencing results confirmed that the cloning process was successful.
Discussion: Genome editing technology has been highlighted in bird species due to their significant potential for economic production and creating models for biological research. The application of CRISPR gene editing technology in avian research has yielded promising advancements, particularly in the modification of somatic cells and tissues in birds. By precisely targeting primordial germ cells (PGCs), researchers can implement specific genetic modifications within the chick genome, thereby facilitating the development of genetically edited chicks. Numerous studies have investigated the role of the PDX-1 gene in gene therapy using the CRISPR/Cas9 system across various organisms. However, this study is the first to design and produce a recombinant construct containing the chicken PDX1 gene using a lentiviral vector with the CRISPR/Cas9 construct. This advancement aims to create a practical research platform for future studies by enabling the direct transfer of this recombinant construct into chick embryos and cells.
Conclusion: The chicken PDX-1 gene was cloned into a lentiviral vector containing the CRISPR/Cas9 construct. The recombinant vector LentiCRISPRv2GFP-sgPDX1 was successfully obtained. The gene construct developed in this study has significant potential for investigating gene function and for use in gene therapy. Consequently, utilizing this construct may enable the creation of an animal model in chicken embryos featuring defects, including an empty organ or tissue cavity. Such a model will allow for the investigation and treatment of the defects using specific drugs and cell lines.