224 CONSTRUCTION OF GENE TARGETING VECTORS FOR RAG-1 AND RAG-2

Gene targeting in mammalian cells has become a routine technique and is currently used to study gene function, create biomedical models, and generate potential tissue sources for xenotransplantation. Severe combined immunodeficiency (SCID) is a condition characterised by the absence of T cells and a lack of B cell function. Severe combined immunodeficiency affects ~1 out of every 100 000 infants. Autosomal recessive SCID can occur due to a mutation within the recombination activating genes (RAG-1/RAG-2) that play a role in recombination of immunoglobulins and T-cell receptors. Gene targeting has been used to create mouse models to study the effects of a RAG-1 or RAG-2 deficiency on the immune system. In 1992, Mombaerts et al. generated a homozygous mouse model of RAG-1 deficiency, whereas Shinkai et al. generated a homozygous mouse model of RAG-2. Both models resulted in the absence of mature T or B lymphocytes; which was concluded to be due to the lack of the ability to initiate the V(D)J recombination process. Because of the anatomical and physiological similarities between humans and pigs, a swine model of both RAG-1 and RAG-2 deficiency would have utility. We hypothesise that disruption of RAG-1, RAG-2, or both in swine will result in a SCID phenotype. A first step in the creation of a swine SCID model is to assemble targeting vectors. The objective of this work was to construct targeting vectors. To accomplish this goal, genomic DNA from porcine fetal fibroblasts was used to amplify a 6840-bp PCR product including the porcine RAG-1 gene. This fragment was cloned into TOPO pCR-XL (Invitrogen, Carlsbad, CA, USA). So that a mammalian G418 resistance cassette could be used for selection of targeting events, this plasmid was modified to remove the endogenous AphII gene (provides G418 resistance). The pKW4 contains LoxP (locus of X-ing over) sites that flank a G418 resistance cassette (based on mammalian codon usage), which is driven by a phosphoglycerate kinase (PGK) promoter (Lorson et al. 2011). This cassette was inserted into the RAG-1 gene to create the targeting construct pAB6. For RAG2, a 9466-bp PCR product i ncluding the RAG-2 gene was amplified and cloned into TOPO pCR-Blunt II (Invitrogen). The LoxP flanked G418 resistance cassette from pKW4 was inserted into the second exon of the RAG-2 gene sequence, creating the targeting construct pAB13. Further, diagnostic screening strategies were developed and validated to discriminate gene-targeting events from random integration. We report here 2 targeting vectors and validated screening methods for gene targeting in porcine fetal fibroblasts that have been validated for cloning. These vectors will be applied toward an effort to create a porcine SCID model. The implications of such a model include evaluation of basic immune function, evaluation of the innate immune system in vaccine efficacy, and organ transplantation.

326 CONSTRUCTION OF A SPLICING-DEPENDENT SELECTABLE MARKER FOR GENE TARGETTING

Gene targeting in mammalian cells plays a crucial role in biotechnology. These experiments are characterised by low rates of homologous recombination and high rates of random integration. Therefore, many fibroblast colonies must be screened to identify a targeting event. To dramatically reduce the survival of random integration events, we have developed a splicing-dependent selectable marker strategy by introducing a mutation in a codon-optimized G418 resistance gene (mNeo). This mutation could be corrected upon homologous recombination. Since the C-terminal region of aminoglycoside phosphotransferase (AphII, Neo/Kan resistance) participates in formation of the active site of this enzyme, we hypothesised that addition of even one amino acid at the C-terminus would render this protein non-functional. To test this hypothesis, a mutation was introduced in an E. coli AphII expression vector that converted the stop codon of AphII to tryptophan (X265W, TGA > TGGTAA). This mutation was confirmed to inactivate AphII by independently characterising the G418 and Kanamycin resistance (or lack thereof) provided by the X265W mutation. To evaluate this mutation in mammalian cells, two intronless mammalian expression vectors were constructed that differed by the presence or absence of the X265W mutation. G418 resistance was only provided by the wildtype sequence, thus confirming that X265W inactivates AphII in mammalian cells. An identical mutation was then introduced into a eukaryotic expression vector based on mNEO. Further, the sequence was extended to create a 5′ intron splice site (TGA > TGGTAAGAGTT). This region was designed to direct splicing between the first and second G residues thus removing the G in the third position of the W codon. The 3′ intron splice sites was then designed to provide an A residue as the first base of the next exon so that successful splicing would correct the mutation by recreating an appropriately positioned stop codon (TGA). To evaluate this strategy in mammalian cells, two plasmids were constructed that harbored the X265W mutation embedded at the 5′ splice site of a downstream intron. In one plasmid (pSC3-G) the first base of the downstream exon begins with a G residue resulting in inactivation of AphII. In the other plasmid (pSC2-A), the first base of the downstream exon begisn with an A residue forming a stop codon that allows for active, wildtype AphII. These plasmids were transfected into porcine fetal fibroblasts and subjected to selection with G418. A positive control plasmid and pSC2-A produced colonies that were too numerous to count. A negative control plasmid and pSC3-G produced no colonies. It can be concluded that the X265W mutation can be corrected by splicing to an exon that begins with an A residue. This splicing-dependent selectable marker may prove useful in gene targeting experiments when the site of modification is followed by an exon that begins with an A.

Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosaβgeo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5′ rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF–treated and –infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACAwas recently defined as a coactivator of c-jun–mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34+ cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis.

Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosaβgeo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5′ rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF–treated and –infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACAwas recently defined as a coactivator of c-jun–mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34+ cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis.