bse-tester
Well-known member
This going to be somewhat of a boring read to most, but it is truly something worth reading - scary also!!!!
This document adresses the genetic sequencing and gene targting being done by Hematech - in between beer production and sampling - and appears to be something they consider quite serious if not the wave of the future. Read on - but pack a lunch, it is consuming!!!
This document adresses the genetic sequencing and gene targting being done by Hematech - in between beer production and sampling - and appears to be something they consider quite serious if not the wave of the future. Read on - but pack a lunch, it is consuming!!!
Gene targeting is accomplished in the mouse using embryonic stem cells; however, in other species, gene targeting has only been successful using primary somatic cells followed by embryonic cloning. Gene targeting in somatic cells is a challenge, compared to mouse embryonic stem cells; consequently, there have been few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report on a sequential gene targeting system for primary fibroblast cells that we used to knockout both alleles of a silent gene, the bovine immunoglobulin (bIghm) gene, and produce both heterozygous and homozygous knockout calves. Furthermore, bIghm targeting was followed by sequential knockout targeting of both alleles of a gene that is active in fibroblasts, the bovine prion protein (bPrnp) gene, in the same genetic line, to produce double homozygous knockout fetuses. The sequential gene targeting system we used alleviates the necessity of germline-transmission for complex genetic modifications and should be broadly applicable for gene-functional analysis, and for biomedical and agricultural applications.
Gene targeting by homologous recombination is a powerful method of specifically modifying a gene of interest and has been used extensively for gene functional analysis in mice1-3. Gene targeting is accomplished in the mouse using embryonic stem (ES) cells; however, for essentially all other species, ES cells, suitable for gene targeting, are not available. In the few reports on non-murine mammalian species, gene targeting was done in primary somatic cells followed by embryonic cloning4-7, which, in some instances, were then used to produce cloned offspring. Gene targeting in primary somatic cells is a challenge8-12 because somatic cells have a relatively short lifespan, which limits selection of properly targeted cell colonies, and have a low frequency of homologous recombination11, compared to mouse ES cells. Because of these limitations, success in somatic cell gene targeting has only been achieved for a couple of genes that were transcriptionally active in the cell line used for targeting and only in sheep and pig. Transcriptionally active genes are more amenable to gene targeting, compared to silent genes, because they have a higher frequency of homologous recombination5, 8 and selection of correctly targeted cells can be easily done by having the targeted gene promoter drive expression of a selection marker; so-called "promoter-less" positive selection4-7, which severely limits its application for transcriptionally silent genes in the somatic cells.
To fully evaluate the consequences of a genetic modification, both alleles of the gene must be targeted. In mice, this is generally done by breeding from heterozygous knockout (KO) founders to produce a homozygous KO inbred line. However, breeding to homozygosity is a severe impediment in species that have a long generation interval, such as cows, sheep and pigs, and that are negatively impacted by the consequences of inbreeding. In pigs, two innovative approaches have been used to circumvent the long generation interval and low rate of homologous recombination for targeting the second allele of the (1, 3)-galactocyltransferase gene. Heterozygous KO fibroblasts were selected in vitro for the lack of the enzymatic activity resulting either from a spontaneous point mutation in the second allele of the gene13 or from mitotic recombinants14. Unfortunately, these approaches are neither useful for silent genes nor widely applicable for active genes.
In this study we developed a broadly applicable and rapid method for generating multiple gene targeting events in cattle. The method consisted of sequential application of gene targeting by homologous recombination and rejuvenation of cell lines by production of cloned fetuses (Fig. 1). This procedure was used to demonstrate the first successful targeting of a transcriptionally silent gene and production of both heterozygous and homozygous KO calves. Furthermore, a second gene was targeted, resulting in double homozygous KO bovine fetuses and its cell lines.
RESULTS
Targeting of the first allele of the bIghm gene in primary fibroblasts
The gene we chose to target was bIghm gene, which is trancriptionally silent in fibroblasts. This gene was characterized in a male Holstein fetal fibroblast cell line (6939) to identify a polymorphic marker DNA sequence, outside the KO vector sequence, which could be used to distinguish the two alleles (Fig. 2a; allele A and allele B as indicated). The first KO vector was constructed using bIghm genomic fragments from around the constant exon 2 region, which was derived from a non-isogenic Holstein genomic library. The KO vector used for targeting the first allele contained a diphtheria toxin A (DT-A) gene15 as a negative selection marker, a puro selection marker driven by a mouse PGK promoter, flanked by loxP sequences and followed by a transcriptional and translational STOP16 cassette (pBCKOpuro, Fig. 2a). Fetal fibroblasts from cell line 6939 were electroporated with the first KO vector to produce 446 wells resistant to puromycin. Wells were split on day 14 and half of the cells were used for screening by polymerase chain reaction (PCR) (primer pairs; puroF2 x puroR2, Fig. 2a) to identify wells containing correctly targeted cells. Initially, six wells were positive by PCR. To exclude false positive wells, all of the PCR products were subjected to bi-directional sequencing analysis with the puroF2 and puroR2 primers. Two wells (0.45%; 147, 384) were identified to be correctly targeted and contain heterozygous bIghm KO (bIghm) cells. Based on polymorphic differences identified by sequence analysis, the KO vector was integrated into allele A in well 384 and into allele B in well 147.
Generation of bIghm fetuses and calves
The remaining cells from the two wells were used for embryonic cloning to generate fetuses and rejuvenate the cell lines. Pregnancy rate at 40 days of gestation was 50% (15/30; two embryos per recipient, Table 1) and at 60 days of gestation, six fetuses were collected and fibroblasts were re-established. Three of six fetuses (2184-1, 2184-2 and 3287) were bIghm (Fig. 2b) as confirmed by the PCR (primer pairs; puroF2 x puroR2) and sequence analysis. Non-targeted fetuses likely resulted either from non-targeted cells that co-existed with the targeted cells in the wells, or from loss of the transgene due to lack of selection pressure during fetal development. Both fetuses 2184-1 and 2184-2 were derived from well 384 where the KO vector was integrated into allele A, and fetus 3287 was from well 147 where the KO vector was integrated into allele B. Cloned, bIghm embryos produced from all three regenerated cell lines were transferred to 153 recipients to produce 13 (8 %, Table 1) healthy bIghm calves, which were confirmed by the PCR (Fig. 2c) and sequence analyses (data not shown).
Targeting of the second allele of the bIghm gene
To target the second allele of bIghm, a second KO vector was prepared in which the puro selection marker was replaced with a neo gene driven by an ST (SV40 promoter and thymidine kinase enhancer) promoter. In attempting to target the second allele of a gene, there is the possibility that the targeting vector will undergo homologous recombination with the integrated targeting vector, resulting in replacement of the KO vector in the previously targeted allele, rather than disruption of the intact allele. This could be a significant problem if the first targeting vector has a strong bias for one allele. This was not observed with our first, non-isogenic, KO vector, indicating either that the two alleles had similar sequences or that polymorphisms had an equal effect on targeting efficiency. We assumed the latter and determined if the frequency of targeting of allele A could be enhanced by constructing a second KO vector in which the short homologous arm was replaced with a PCR derived sequence amplified directly from allele A of the cell line 6939 (this vector was designated pBCNKOneo).
All three bIghm cell lines (2184-1 and 2184-2, targeted in allele A; 3287, targeted in allele B) were used for targeting with the second KO vector (Fig. 2a). In cell lines 2184-1 and 2184-2, a total of 1,211 wells, resistant to G418, were screened by PCR (primer pairs; neoF3 x neoR3; Fig 2a) followed by sequence analysis. Five wells were positive and, in two (0.17%), the vector was integrated into the intact allele B, producing homozygous KO (bIghm) cells, and in three wells the targeting vector in allele A was replaced. In cell line 3287, 569 wells, resistant to G418, were screened by PCR (primer pairs; neoF3 x neoR3; Fig 2a) followed by sequence analysis. Seven wells were positive and, in six (1.1%), the vector was integrated into the intact allele A producing bIghm cells and, in one well, the targeting vector in allele B was replaced. Overall the vector had a bias of 6:1 for intact allele A to B and was more efficient for homozygous targeting when used with cell line 3287 where allele B was firstly targeted, as expected.
Generation of bIghm fetuses and calves
Two bIghm wells (76 and 91), derived from cell line 3287, were selected for embryonic cloning to generate fetuses and rejuvenate the cell lines. Overall pregnancy rate for bIghm fetuses at 40 to 50 days of gestation was 45% (40/89, Table 1). At 45 days of gestation, 5 fetuses derived from well 76 and 15 fetuses from well 91 were collected and evaluated. All five from well 76 (Fig. 2d) and three out of the 15 from well 91 (not shown) were positive in PCR specific for the first and second targeting events (primer pairs; puroF2 x puroR2 and neoF3 x neoR3). PCR results were confirmed by sequence analyses and negative PCR17 (primer pairs; bCf x bCr; Fig. 2a) for the wild type alleles (Fig. 2d). Confirmation of a functional knockout was obtained by generation of 90 day fetuses from regenerated bIghm fibroblasts and evaluation of bIghm gene expression in spleen cells. The absence of expression was confirmed by RT-PCR (primers pairs; bCf x bCr, Fig. 2e). Cloned embryos were made from 5 bIghm cell lines and were transferred to recipients for development to term. Eight calves (6 %, Table 1) were born recently and were confirmed to be bIghm by PCR (Fig. 2f) and sequence analyses (data not shown), verifying that sequential gene targeting and successive rounds of cell rejuvenation are compatible with full term development of healthy homozygous KO calves (Fig. 2g).
Cre-mediated excision of floxed neo and puro genes in the bIghm fibroblasts
Sequential gene targeting requires a strategy for antibiotic selection of a newly integrated targeting vector in a cell line that already contains one or multiple antibiotic selection markers. The simplest approach is to use a different selection marker gene for each targeting event; however, this approach limits the number of targeting events that may be performed in a cell line. Another approach is to remove the selection markers using a Cre-loxP recombination system, as has been done in murine embryonic stem cells18.
Unexpectedly, in our regenerated bIghm targeted fibroblasts, the selection marker genes were not expressed, likely because reprogramming of fibroblasts following embryonic cloning resulted in silencing of the newly integrated sequence as part of the silent bIghm locus. Although selection marker removal was not necessary for further targeting in our bIghm fibroblasts, we evaluated the possibility of removing the selection markers by transfection with a Cre recombinase expression plasmid. Because the intention was for transient expression of Cre recombinase, a closed circular plasmid was used and antibiotic selection was restricted to the first three days of culture. Bovine Ighm cell line 4658 was used for transfection and 24 selected wells were evaluated by PCR for excision of the antibiotic selection genes from the targeted alleles (Fig. 3a). Multiple wells showed evidence of excision of both puro and neo genes and one was chosen for fetal cloning and regeneration of cell lines. Pregnancy rate at 40 to 50 days of gestation was 35% (21/60, Table 1). Five fetuses were recovered and all had both selection markers removed (Fig. 3b), however, the Cre recombinase plasmid integrated into the genome in all fetuses, except fetus 1404 (data not shown). These results indicate that Cre-loxP recombination can be used to remove selection markers in somatic cells; however, routine use in this system will require improvements to reduce integration frequency of Cre-expression plasmid.
Targeting of the first allele of the bPrnp gene in bIghm fibroblasts
To evaluate the possibility of sequentially targeting a second gene, Cre-excised bIghm (Cre/bIghm) fibroblasts (cell line 1404) were subjected to a third round of targeting to disrupt the bPrnp gene. This gene was first characterized to identify a polymorphic sequence, outside the KO vector sequence, to distinguish the two alleles (Fig. 4a; allele C and allele D as indicated). The vector comprised non-isogenic sequences derived from the region around exon 3 of the bPrnp gene and the DT-A gene, the neo selection marker driven by the ST promoter, flanked by loxP sequences and followed by the STOP cassette (pBPrP(H)KOneo, Fig. 4a). Cells were transfected with the third KO vector and 203 G418-resistant wells were screened by PCR. Thirteen (6.4%) wells with cells showing a heterozygous KO in the bPrnp gene on Cre/bIghm background (Cre/bIghm/bPrnp) were identified (primer pairs; neoF7 x neoR7; Fig. 4a; data not shown). Sequence analysis showed that the third KO vector was integrated into allele C of the bPrnp gene in all the positive wells. Some wells were used for cloning to generate 28 pregnancies at 45 days of gestation (71%, Table 1). Five fetuses were collected and all were positive for targeting at allele C of the bPrnp gene as confirmed by PCR (Fig. 4b; primer pairs; neoF7 x neoR7) and sequencing analyses (not shown). Furthermore, no amplification was detected following negative bIghm PCR, as expected (Fig. 4b; primer pairs; bCf x bCr). Targeting efficiency for bPrnp, a gene that is transcriptionally active in bovine fibroblasts, was substantially higher than for bIghm (6.4% vs. 0.63%, respectively), a gene that is not expressed in fibroblast cells.
Targeting of the second allele of the bPrnp gene in Cre/bIghm/bPrnp fibroblasts.
To examine the feasibility of quadruple targeting to produce double homozygous KO fetuses and cell lines, the triple targeted cell line (8443, Cre/bIghm/bPrnp) was transfected with a fourth KO vector for the remaining allele of the bPrnp gene. The vector was constructed by replacing the neo gene with the puro gene (pBPrP(H)KOpuro, Fig. 4a) in the bPrnp targeting vector used for the first allele. As a result of selection and PCR screening (primer pairs; puroF14 x puroR14, Fig. 4a), 17 (5.2%) wells were found to contain targeted cells.
Sequence analysis confirmed that the fourth KO vector was integrated into allele D of the bPrnp gene, creating double homozygous KO (Cre/bIghm/bPrnp) cells, in all positive wells, except one, in which the targeted sequence in allele C was replaced. Cells from Cre/bIghm/bPrnp positive wells were used for cloning to produce fetuses. The pregnancy rate derived from these embryos at 45 days of gestation was 68% (Table 1). All 18 fetuses that were collected were Cre/bIghm/bPrnp as confirmed by positive PCR analysis using the targeting event-specific primer pairs, puroF14 x puroR14 and neoF7 x neoR7 (Fig. 4c).
Sequencing analyses confirmed integration of the third (neo) and fourth (puro) bPrnp targeting vectors into alleles C and D, respectively. Furthermore, we performed a negative PCR analysis to confirm the absence of wild type bPrnp alleles (primer pairs; BPrPex3F x BPrPex3R, Fig. 4c) and bIghm alleles (primer pairs; bCf x bCr, data not shown) and, as expected, all four KOs were confirmed. To evaluate bPrnp mRNA expression, fibroblasts from a bIghm, a Cre/bIghm/bPrnp and Cre/bIghm/bPrnp fetuses were examined by RT-PCR. Functional disruption of bPrnp gene expression was confirmed (Fig. 4d). These results indicate that multiple rounds of gene targeting, both for transcriptionally active and silent genes, were readily accomplished in a single somatic cell line using a cell rejuvenation approach.
DISCUSSION
In this study we demonstrate, for the first time, a sequential gene targeting strategy for primary somatic cells, which can be used for targeting multiple alleles of a gene or for targeting multiple genes. The system proved effective for targeting both transcriptionally silent and active genes, demonstrating broad application, and was compatible with development of healthy calves through at least two rounds of gene targeting. Furthermore, there was no indication that additional rounds of gene targeting compromised development of cloned embryos, as judged from pregnancy rates at 45-60 days of gestation (Table 1). Pregnancies with the double homozygous knockout fetuses are in progress and pregnancy rates are consistent with the results obtained in this study.
One advantage of the sequential gene targeting system is that the time required to produce an animal with multiple genetic modifications is greatly reduced compared to traditional breeding strategies. With sequential gene targeting, each targeting event required approximately 2.5 months from transfection to establishment of regenerated cell lines, therefore, homozygous targeted calves could be made in 14 months (5 months for targeting two alleles and 9 months gestation) and double homozygous targeted calves, including Cre-mediated excision of selection genes, could be made in 21.5 months (Fig. 1). In contrast, for cattle, breeding a heterozygous founder to produce homozygous calves would require approximately 5 years and generation of double homozygotes from two heterozygous founders is impractical.
Several factors were important for maximizing targeting efficiency and for successful production of rejuvenated cell lines and calves. Overall, frequency of homologous recombination at each targeting step was sufficiently high (0.4 to 6.4%) to produce at least a couple of targeted colonies out of about 500 selected colonies that were screened by PCR in each experiment. The efficiency might be attributed to several conditions that were optimized specifically for bovine fibroblast targeting. To maximize expression of the positive selection marker genes we chose two, ubiquitously active promoters; ST (SV40 promoter and thymidine kinase, tk enhancer) and murine PGK promoters. For negative selection against random integrations, the diphtheria toxin A (DT-A) gene was used, instead of the typical herpes simplex virus (HSV)-tk negative selection19, to avoid exposing the cells to a second selective drug. The DT-A gene was likely important because homologous recombinants were not detected when negative selection was not used in preliminary studies. Efficiency was also maximized by constructing vectors comprising contiguous regions of homology within the targeted gene loci and not creating a deletion as a result of homologous recombination5. Rather than deleting sequence to disrupt the targeted genes, we inserted both transcriptional and translational termination sequences. In addition to optimizing the targeting vector, we also optimized the transfection, selection and cloning procedures. Low passage fetal fibroblasts were used for all transfections. Electroporation conditions were optimized to produce about 1,000 drug-resistant colonies per 1 x 107 transfected cells, when not using negative selection. Cloning from selected cell colonies was initiated the day following PCR screening to minimize progress towards senescence. Finally, cloning was done using a modified system to facilitate reprogramming of the donor cells20.
Using this sequential targeting strategy, complex genetic modifications, in large animal species, are, not only, feasible but relatively straightforward and should be useful for many applications. Multiple genes targeting in large animals may be useful for production of novel models for human disease, for production of various therapeutic proteins, for production of organs or tissues for transplantation into human patients and for improving the efficiency of agricultural production. Gene targeting has many useful applications in science, medicine and industry and may be one of the most useful applications of somatic cell cloning technology. Currently gene targeting using ES cells has only been successful in mice; however, somatic cell cloning has been successful for many species21-25. The results obtained in this study indicate that complex genetic modifications can now be readily made for a wide variety of genes in many species.
METHODS
Construction of KO vectors. A bovine genomic fragment around exon 2 of the bIghm constant region locus was obtained from non-isogenic Holstein genomic library by probing with a 32P-labeled PCR fragment. One genomic clone was analyzed further by restriction mapping. The 7.2 kb of Bgl II-Xho I genomic fragment (5' homologous arm) and 2.0 kb of Bam HI-Bgl II fragment (3' homologous arm) around the exon 2 were subcloned into pBluescript II SK(-) (Stratagene), and then puro, STOP cassettes (pBS302, Stratagene) and DT-A (diphtheria toxin A) genes were inserted {pBCKOpuro vector}. For construction of the second targeting vector, genomic PCR was performed from cell line 6939. After digestion with Bam HI-Bgl II, the fragment replaced the 3' short arm of the pBCKOpuro vector. By sequencing, the Bam HI-Bgl II fragment was confirmed to be amplified from "allele A". The puro gene was replaced with a neo gene {pBCKOneo vector}.
Bovine genomic fragment around exon 3 of bPrnp locus was obtained by screening of the same Holstein genomic phage library with a 32P-labeled DNA fragment amplified by PCR. One genomic clone was analyzed further by restriction mapping. The 8.3 kb of Bam HI genomic fragment (3' homologous arm) and 1.2 kb of Bam HI-Bgl II fragment (5' homologous arm) containing the exon 3 were subcloned into pBluescript II SK(-), and then both neo and STOP cassettes were inserted at the Bam HI site, which is behind the initial ATG codon. The DT-A gene was also subcloned {pBPrP(H)KOneo vector}. Similarly, another KO vector containing the puro gene was constructed {pBPrP(H)KOpuro vector}. Primer sequences are available on request.
Cell culture and transfection. Holstein fetal male fibroblasts were cultured as previously described26 and electroporated with 30 g of each targeting vector at 550 V and 50 F by using a GenePulser II (Bio-rad). After 48 hours, the cells were selected under 500 g/ml of G418 or 1 g/ml of puromycin for two weeks and the drug-resistant colonies were picked and transferred to replica plates; one for genomic DNA extraction (24-well plates) and the other for embryonic cloning (48-well plates).
Genomic PCR analyses. From the replica 24-well plates, fetus or ear biopsy genomic DNA from calves, was extracted using a Puregene DNA extraction kit (GentraSystem). To identify each homologous recombination event that occurred at bIghm locus, puroF2, puroR2, neoF3 and neoR3 primer pairs (Fig. 2a) were used. PCR was performed in 30 cycles comprising 98C-10s, 68C-8min. For negative PCR, BCf and BCr primer pairs (Fig. 2a) were used in 40 cycles of PCR composed of 98C-10s, 62C-30s, 72C-1min. In the case of the bPrnp locus, neoF7, neoR7, puroF14 and puroR14 primer pairs were used (Fig. 4a). PCR was performed in 30 cycles comprising 98C-10s, 68C-5min. For negative PCR, BPrPexF and BPrPexR primer pairs (Fig. 4a) were used in the 40 cycles of PCR composed of 98C-10s, 62C-30s, 72C-1min. To detect the Cre-mediated excision, PCR was carried out with CreExF and CreExR primer pair (Fig. 3a) in 40 cycles of PCR composed of 98C-10s, 68C-7min. All the PCR products were run on 0.8% agarose gels. Primer sequences are available on request.
Sequencing analysis of the PCR products. To confirm whether homologous recombination correctly occurred at each targeting step, the PCR products amplified above were sequenced. The PCR products were purified through CHROMA SPIN-TE400 column (BD Biosciences Clontech) and sent to ACGT Inc. (Wheeling, IL) to sequence. Sequence was bi-directionally done both with forward and reverse primers which were used for PCR. The allele into which each KO vector was integrated was determined by polymorphisms in the sequence of the PCR products.
Embryonic cloning. Cloned fetuses and calves were produced using as described previously20. In vitro matured oocytes were enucleated at 20 h post maturation. Correctly targeted clones were permeabilized by incubation of about 50-100,000 cells in suspension with 31.2 U Streptolysin O (SLO; Sigma) in 100 μl HBSS for 30 min in a 37°C H2O bath. Permeabilized cells were sedimented, washed and incubated with 40 μl mitotic extract containing an ATP generating system (1 mM ATP, 10 mM creatine phosphate and 25 μg/ml creatine kinase) for 30 min at 38°C. At the end of the incubation, the reaction mix was diluted, sedimented and washed. These cells were fused to enucleated oocytes, activated at 28 h post maturation with 5 μM calcium ionophore for 4 min followed by 10 μg/ml cycloheximide and 2.5 μg/ml cytochalasin D for 5 h. After activation, embryos were washed and co-cultured with mouse fetal fibroblasts to the blastocyst stage in vitro. Grade 1 and 2 blastocysts were selected and transferred into synchronized recipients. All animal work was done following a protocol approved by the Transova Genetics Institutional Animal Care and Use Committee.
RT-PCR. RNA was extracted from spleen of wild type (6939) and bIghm fetuses using an RNeasy mini kit (Qiagen) and first strand cDNA synthesis was done using the superscript first strand synthesis system for RT-PCR (Invitrogen). PCR was done using BCf and BCr primers in 40 cycles of PCR composed of 98C-10s, 62C-30s, 72C, 1min. RNA was also extracted from 4658 (bIghm), 8443 (bIghm/bPrnp) and double homozygous KO (bIghm/bPrnp) fibroblasts and first strand cDNA synthesis was done as above. PCR was done by using PrPmF3 and PrPmR3 primers in 40 cycles of 98C-10s, 62C-30s, 72C-1min. For detection of bovine -actin mRNA expression, bBAF and bBAR primers were used in the same PCR condition (data not shown). To exclude the possibility of genomic DNA contamination, another RT-PCR was performed without reverse-transcriptase (data not shown). The PCR products were ran on 0.8% agarose gel. Primer sequences are available on request.
ACKNOWLEDGEMENTS
We thank J. Pommer, J. Koster, J. Molina and D. Faber for their assistance in embryo transfer, fetal recovery, calf delivery and sample collection. We also thank M. Nichols, J. Griffin, M. Bien, T. King, M. Ahlers, R. Paulson, S. Viet and C. Voss for their assistance in gene targeting and nuclear transplantation.
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Figure 1. Diagram of procedure for sequential gene targeting in bovine primary fibroblasts. Holstein fetal fibroblasts (6939) were targeted, then wells containing targeted cells were selected and cloned to generate bIghm fetuses. The bIghm cell line (3287) was then used for production of calves and for targeting the second allele of bIghm. Once again, cells were selected and regenerated by production of fetuses. Fetuses were harvested for production of bIghm cell lines, bIghm gene expression analysis and production of calves. A bIghm cell line (4658) was transfected with a Cre-recombinase expression plasmid to remove both neo and puro genes simultaneously, followed by a third round of embryonic cloning to generate cloned fetuses and cell lines in which both neo and puro selection marker genes were excised. One Cre-excised bIghm fibroblast cell line (1404) was used for a third round of gene targeting to produce triple targeted, Cre/bIghm/bPrnp fetuses and cell lines. One cell line (8334) was subjected to the fourth round of gene targeting to produce double homozygous KO (Cre/bIghm/bPrnp) fetuses and cell lines and for evaluation of bPrnp gene expression. A representative timeline for each step is indicated.
Figure 2. Sequential targeting of the bIghm gene in primary bovine fibrobalsts. (a) The figure depicts the structure of bIghm constant region locus in cell line 6939, the puro and neo vectors used for the first and second round of targeting, respectively and the genomic PCR assay used for the first and second targeting events. The targeting vectors were composed of a 5' homologous arm (7.2 kb), a 3' homologous arm (2.0 kb), STOP cassette containing transcriptional and translational stop sequence, DT-A (diphtheria toxin A gene) and floxed puro or neo gene. The vectors were designed to insert the knockout cassette into exon 2 of the bIghm constant region locus. In fibroblasts 6939, polymorphic sequences were found to distinguish allele A and allele B, as indicated. Primer pairs, puroF2 x puroR2 were used to identify the first targeting event. PCR and sequencing product showed that the first puro KO vector was integrated into allele B in cell line 3287 and in allele A in cell lines 2184-1 and 2184-2 (not shown), based on the polymorphic sequences presented in the PCR product. Primer pairs, neoF3 x neoR3 were used to identify the neo second targeting event at allele A to produce cell line 4685. BCf x BCr is a primer pair used to confirm the absence of wild type alleles after the second targeting. The primers did not amplify the targeted alleles because of the added length of sequence from the integrated STOP cassettes. (b) Identification of bIghm fetuses by genomic PCR with puroF2 x puroR2 primers. N is a negative control (mixture of the 1st KO vector and genomic DNA of cell line 6939) and P is a positive control (mixture of about 104 copies/l of plasmid DNA covering puroF2-puroR2 region and genomic DNA of cell line 6939). Cell lines 2184-1, 2184-2 and 3287 were bIghm. (c) Genotyping of bIghm calves by genomic PCR with puroF2 x puroR2 primers. N is a negative control (mixture of the 1st KO vector and genomic DNA of cell line 6939) and P is a positive control (mixture of about 104 copies/l of plasmid DNA covering puroF2-puroR2 region and genomic DNA of cell line 6939). Out of 13 bIghm calves born, five were genotyped. All of them were positive to the 1st targeting event. (d) Identification of bIghm fetuses and fibroblasts by genomic PCR with puroF2 x puroR2, neoF3 x neoR3 and BCf x BCr primers. "P" is a positive control (mixture of about 104 copies/l of plasmid DNA covering either puroF2-puroR2 or neoF3-neoR3 region and genomic DNA of cell line 6939). "N" is a negative control (mixture of either the 1st KO or 2nd KO vector and genomic DNA of cell line 6939) and 6939 is the original fibroblast cell line. Cell lines 4658, 3655, 5109, 5139 and 4554 were positive for the targeting events both at allele A (neo-targeting) and B (puro-targeting), but negative for wild type alleles. (e) RT-PCR analysis on bIghm gene expression in mRNA extracted from spleen in day 90 fetuses. Clear expression from a positive control "P" (commercially available polyA+ bovine spleen RNA) and the wild type (6939) fetuses (#1, #2), but not from bIghm fetuses, was detected. (f) Genotyping of bIghm calves by genomic PCR with puroF2 x puroR2, neoF3 x neoR3 and BCf x BCr primers. N is a negative control (mixture of either the 1st KO or 2nd KO vector and genomic DNA of cell line 6939) and P is a positive control (mixture of about 104 copies/l of plasmid DNA covering either puroF2-puroR2 or neoF3-neoR3 region and genomic DNA of cell line 6939). Out of 8 bIghm calves born, two calves were genotyped and were positive for targeting events at both allele B and A of bIghm gene, but were negative for the wild type alleles.
Figure 3. Removal of both neo and puro genes by Cre-loxP system. (a) The figure depicts the structure of alleles of bIghm cell line 4658 and the genomic PCR assay for Cre-loxP mediated removal of selection marker genes. Amplification from primer pairs, CreExF x CreExR, results in a 2.5 kb fragment from the puro targeted allele, a 4.3 kb from the neo targeted allele or a short 0.4 kb fragment when both selection marker genes were excised. (b) Identification of Cre/bIghm fetuses and fibroblasts by genomic PCR with CreExF x CreExR primers. In cell line 4658, prior to introduction of Cre, 2.5 kb (puro) and 4.3 kb (neo) PCR products are detected. In the five Cre/bIghm fetuses and cell lines, these bands completely disappear and, instead, a 0.4 kb (without puro and neo) band is detected.
Figure 4. Sequential targeting of the bPrnp gene in the bIghm¬ fibroblasts. (a) The figure illustrates the structure of bPrnp locus in Cre/bIghm cell line (1404), the neo and puro vectors used for the third and fourth round of targeting, respectively and the genomic PCR assay for the third and fourth targeting events. The vector was composed of a 5' homologous arm (1.2 kb), a 3' homologous arm (8.3 kb), STOP cassette, DT-A gene and floxed neo or puro gene. The vectors were designed to insert the knockout cassette just behind its initial ATG codon located in exon 3 of the bPrnp locus. A single base pair polymorphism was found between allele C and allele D as indicated. Primer pair, neoF7 x neoR7, was designed to show the neo targeting event and include the polymorphic base. PCR and sequencing products showed that the third neo vector was integrated into allele C. Primer pairs, puroF14 x puroR14 were used to identify the fourth puro targeting event at allele D. BPrPex3F x BPrPex3R is a primer pair used to confirm the absence of wild type alleles after the fourth targeting. The primers would not amplify sequence from the targeted alleles because of the disruption of annealing sites of BPrPex3F primer on the both alleles of bPrnp gene caused by the homozygous insertion. (b) Identification of the triple targeted fetuses and fibroblast cell lines (bIghm/bPrnp) by genomic PCR with positive bPrnp primer pair, neoF7 x neoR7, and negative bIghm primer pair, BCf x BCr. P is a positive control (mixture of about 104 copies/l of plasmid DNA covering neoF7-neoR7 region and genomic DNA of cell line 6939) and cell line 1404 is a negative control. Cell lines derived from fetuses 8103, 1661, 8375, 8112 and 8443 were positive for the bPrnp targeting and negative for wild type bIghm alleles, demonstrating that they were triple targeted cell lines (bIghm/bPrnp). (c) Identification of double homozygous KO (bIghm/bPrnp) fetuses and fibroblasts by genomic PCR with puroF14 x puroR14, neoF7 x neoR7 and BPrPex3F x BPrPex3R primers. "P" is a positive control (mixture of about 104 copies/l of plasmid DNA covering either puroF14-puroR14 or neoF7-neoR7 region and genomic DNA of cell line 6939). "N" is a negative control (mixture of either the 3rd KO or 4th KO vector and genomic DNA of cell line 6939) and the bIghm fetal cell line (4658) and the Cre/bIghm/bPrnp cell line (8443) are indicated. Cell line 8454, 8400 and 6397 are three of the double homozygous KO (Cre/bIghm/bPrnp) fetuses. They were positive to the third and fourth targeting events at alleles C (neo-targeting) and D (puro-targeting) of the bPrnp gene, but negative to wild type alleles of the bPrnp gene. (d) RT-PCR analysis on the double homozygous KO (Cre/bIghm/bPrnp) fetuses. To detect bPrnp mRNA, PrPmF3 x PrPmR3 primers were used. Clear expression from the 4658 (bIghm) and the 8443 (Cre/bIghm/bPrnp) fetuses, but no expression from the double homozygous KO (Cre/bIghm/bPrnp) fetuses, was observed.
Table 1. Production of cloned fetuses and calves from bIghm and Prnp targeted fibroblasts
Type of modification Endpointa No of recipients implanted No of pregnancy at 40-45 d (%) No of live calves (%)
bIghm Fetus 30 15 (50) -
bIghm Calf 153 99 (65) 13 (8)
bIghm Fetus 89 40 (45) -
bIghm Calf 137 86 (63) 8 (6)
Cre/bIghm Fetus 60 21 (35) -
Cre/bIghm/bPrnp Fetus 39 28 (71) -
Cre/bIghm/bPrnp Fetus 67 46b (68) -
aFetuses were produced from selected colonies and calves were produced from rejuvenated cryopreserved cell lines. bAfter removing fetuses from 26 pregnant recipients, 15 pregnancies were left to continue to full term and 9 of them were confirmed pregnant at 60 d.
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