The breakpoint position was determined by FISH using Bacterial Artificial Chromosome (BAC) clones obtained from the INRAE @BRIDGe Platform (http://abridge.inra.fr/index.php , 1 April 2020) [27 (link)]. The BACs SBAB-91F6 (located on SSC1) and CH242-4A8 (located on SSC15) were labeled with biotin using the BioPrime DNA Labeling System kit (LIFE TECHNOLOGIES, Carlsbad, CA, USA) and revealed by Alexa 594 conjugated to Streptavidin (MOLECULAR PROBES; Eugene, OR, USA). The BAC CH242-29P21 (SSC15) was labeled by digoxygenin and revealed using FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St. Louis, MO, USA), as previously described [31 (link)].
Alexa 594 conjugated to streptavidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 594 conjugated to Streptavidin is a fluorescently labeled protein complex. Streptavidin is a tetrameric protein that binds strongly to the small molecule biotin. The Alexa 594 dye is attached to the streptavidin, providing a red fluorescent signal that can be detected and visualized.
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Lab products found in correlation
Bioprime dna labeling system kit, by Thermo Fisher Scientific (5 mentions)
Fitc conjugated mouse anti digoxygenin antibodies, by Merck Group (5 mentions)
Bioprime labeling system, by Thermo Fisher Scientific (3 mentions)
Fluorescein isothiocyanate fitc conjugated mouse anti digoxygenin antibodies, by Merck Group (2 mentions)
8 protocols using alexa 594 conjugated to streptavidin
1
Chromosomal Mapping of Porcine Genome
2
Chromosome Painting via Interline-PCR FISH
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Probes for FISH experiments were generated using 50 ng of hybrid DNA as template for interSINE-PCR amplification as described previously [20 (link)]. For each probe, 2 μl of pIRS-PCR amplified hybrid DNA were labeled by random priming with biotin-16-dUTP using the BioPrime labeling system (Invitrogen). The probes were precipitated in the presence of competitor DNA (5 μg of porcine Cot-1 DNA). The DNA pellets were resuspended in 25 μl of hybridization mixture, denaturated and preannealed for 3 h at 37°C to block repetitive sequences. FISH experiments were performed according to [21 (link)]. The probes were detected using Alexa 594 conjugated to Streptavidin (Molecular Probes, Eugene, OR, USA) [20 (link)].
Painted chromosomes or fragments of chromosomes were identified by comparison with G-banded metaphase chromosome pictures taken before hybridization [20 (link)].
Painted chromosomes or fragments of chromosomes were identified by comparison with G-banded metaphase chromosome pictures taken before hybridization [20 (link)].
3
Chromosomal Location Identification via FISH
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After SC analysis, the same cells were subjected to FISH with BAC clones (SBAB-428G6, SBAB-413G8, SBAB-498D8 from the National Institute for Agricultural Research (INRA) BAC library) (Rogel-Gaillard et al. 1999 (link)): one was located in the telomeric region of the SSC1 p arm (labeled with biotin), and two were located on SSC14, one in the telomeric region (labeled with digoxigenin) and the other in the centromeric region (containing porcine endogenous retrovirus (PERV) sequences, differentially labeled with biotin and digoxigenin). These BAC clones were labeled using the BioPrime DNA Labeling System kit (Life Technologies, Carlsbad, CA, USA), and revealed by Alexa 594 conjugated to streptavidin (Molecular Probes, Eugene, OR, USA) and fluorescein isothiocyanate (FITC) conjugated mouse anti-digoxygenin antibodies (Sigma-Aldrich, Saint Louis, MO, USA). FISH signals from the same cells for which SCs had previously been analyzed were captured and evaluated using an Imager Z2 microscope with CytoVision imaging system (Leica Microsystemes, Nanterre, France).
4
Chromosome Organization Analysis by FISH
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After SC analysis, the same cells were subjected to fluorescence in situ hybridization with BAC clones: one was located in the telomeric region of the SSC1 p-arm (labeled with biotin), and one in the centromeric region of SSC15. These BAC clones were labeled using the BioPrime DNA Labeling System kit (LIFE TECHNOLOGIES, Carlsbad, CA, USA), and revealed by Alexa 594 conjugated to Streptavidin (MOLECULAR PROBES, Eugene, OR, USA) and fluorescein isothiocyanate (FITC) conjugated mouse anti-digoxygenin antibodies (SIGMA-ALDRICH, St. Louis, MO, USA). FISH signals from the same cells for which SCs had previously been analyzed were captured and evaluated using an Imager Z2 microscope with the Cytovision imaging system (LEICA MICROSYSTEMES, Nanterre, France).
5
Chromosome Identification via FISH
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FISH experiments were performed on the same slides, according to Massip et al. (2010) [31] , with slight modifications. The 18 autosomal bivalents were identified by 3 successive hybridizations of BAC probe combinations, as presented in Figure 1 . These BAC probes were selected in the telomeric regions of the chromosomes [32] and were obtained from the Biological Resources Center-GADIE (http://www-crb.jouy.inra.fr/ ) [33] . Probes were labeled with biotin or digoxygenin using the bioprime labeling system (Invitrogen) and revealed using Alexa 594 conjugated to Streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
6
BAC Probe Hybridization in Telomeric Regions
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BAC (bacterial artificial chromosome) probes were hybridized on the same slides according to Mary et al. (2014) [8 (link)]. These BAC probes were selected in the telomeric regions of the chromosomes [27 (link)] and were obtained from the Biological Resources Center-GADIE (http://www-crb.jouy.inra.fr/ ) [28 (link)].
Different combinations of probes were used:
Probes were labeled with biotin or digoxygenin using the bioprime labeling system (Invitrogen) and revealed using Alexa 594 conjugated to Streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
Different combinations of probes were used:
Probes were labeled with biotin or digoxygenin using the bioprime labeling system (Invitrogen) and revealed using Alexa 594 conjugated to Streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
7
Cytogenetics of Caprine Polycerate Mutation
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Skin biopsies were sampled from one heterozygous polycerate and one wild-type fetuses. Fibroblast cultures and metaphases were obtained according to (Ducos et al. 2000 (link)). Nucleotide sequences from the segments of caprine chromosomes 2 and 5 involved in the candidate causative mutation were aligned against bovine bacterial artificial chromosome (BAC) end sequences using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed November 4, 2020 ). Two INRA BAC clones (Eggen et al. 2001 (link)) were selected and obtained from the Biological Resources of @BRIDGe facilities (abridge.inrae.fr): INRAb 230B11, targeting the segment deleted on Chr2, and INRAb 348A12, targeting the region of Chr5 that is duplicated and inserted on Chr2. FISH experiments were carried out according to (Yerle et al. 1994 ). The two BACs were labeled with biotin and digoxygenin, respectively, using the BioPrime DNA Labeling System kit (Life Technologies, Carlsbad, CA). Finally, they were revealed by Alexa 594 conjugated to streptavidin (Molecular Probes, Eugene, OR) and FITC conjugated mouse antidigoxygenin antibodies (Sigma, St Louis, MO).
8
Cytogenetic Analysis of Polycerate Goats
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Skin biopsies were sampled from one heterozygous polycerate and one wild-type fetuses.
Fibroblast cultures and metaphases were obtained according to (Ducos et al. 2000) . Nucleotide sequences from the segments of caprine chromosomes 2 and 5 involved in the candidate causative mutation were aligned against bovine bacterial artificial chromosome (BAC) end sequences using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Two INRA BAC clones (Eggen et al. 2001) were selected and obtained from the Biological Resources of @BRIDGe facilities (abridge.inrae.fr): INRAb 230B11, targeting the segment deleted on Chr2, and INRAb 348A12, targeting the region of Chr5 that is duplicated and inserted on Chr2. FISH experiments were carried out according to (Yerle et al. 1994) . The two BACs were labeled with biotin and digoxygenin, respectively, using the BioPrime DNA Labeling System kit (Life Technologies, Carlsbad, CA, USA). Finally, they were revealed by Alexa 594 conjugated to streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
Fibroblast cultures and metaphases were obtained according to (Ducos et al. 2000) . Nucleotide sequences from the segments of caprine chromosomes 2 and 5 involved in the candidate causative mutation were aligned against bovine bacterial artificial chromosome (BAC) end sequences using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Two INRA BAC clones (Eggen et al. 2001) were selected and obtained from the Biological Resources of @BRIDGe facilities (abridge.inrae.fr): INRAb 230B11, targeting the segment deleted on Chr2, and INRAb 348A12, targeting the region of Chr5 that is duplicated and inserted on Chr2. FISH experiments were carried out according to (Yerle et al. 1994) . The two BACs were labeled with biotin and digoxygenin, respectively, using the BioPrime DNA Labeling System kit (Life Technologies, Carlsbad, CA, USA). Finally, they were revealed by Alexa 594 conjugated to streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
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