Mouse Tcim Conditional Knockout Project (CRISPR/Cas9)

https://www.alphaknockout.com

Objective: To create a Tcim conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tcim gene (NCBI Reference Sequence: NM_026931.2 ; Ensembl: ENSMUSG00000056313 ) is located on Mouse chromosome 8. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000052622). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tcim gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-248I3 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit myeloid and lymphoid hyperplasia, an increased number of small- sized red blood cells, increased hematopoietic stem cell number, and enhanced hematopoietic activity.

Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~2227 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Tcim cKO region loxP site

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Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(6318bp) | A(27.9% 1763) | C(19.55% 1235) | T(29.66% 1874) | G(22.89% 1446)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 - 24438897 24441896 3000 browser details YourSeq 195 810 1175 3000 87.3% chr12 + 85148876 85149231 356 browser details YourSeq 174 802 1172 3000 90.7% chr11 - 69869925 69870300 376 browser details YourSeq 172 977 1192 3000 92.7% chr1 + 33784304 33784583 280 browser details YourSeq 167 967 1185 3000 88.6% chr10 + 60702287 60702488 202 browser details YourSeq 164 980 1172 3000 94.2% chr10 + 63088247 63088453 207 browser details YourSeq 160 846 1172 3000 87.0% chr6 + 120253632 120253906 275 browser details YourSeq 160 982 1184 3000 90.4% chr18 + 63958745 63958932 188 browser details YourSeq 159 838 1172 3000 86.9% chr7 - 139282408 139282732 325 browser details YourSeq 159 981 1171 3000 91.3% chr12 + 12926730 12926906 177 browser details YourSeq 158 969 1159 3000 90.9% chr11 + 94110925 94111102 178 browser details YourSeq 158 979 1193 3000 90.7% chr11 + 32741708 32742237 530 browser details YourSeq 156 846 1141 3000 90.8% chr16 - 20503841 20504134 294 browser details YourSeq 155 982 1170 3000 89.4% chr11 - 87257882 87258059 178 browser details YourSeq 155 958 1161 3000 91.5% chr2 + 146251072 146251276 205 browser details YourSeq 154 982 1172 3000 89.7% chr2 - 92999220 92999395 176 browser details YourSeq 153 790 1172 3000 83.8% chr14 - 34550821 34551001 181 browser details YourSeq 152 982 1175 3000 90.0% chr8 - 13948142 13948332 191 browser details YourSeq 152 983 1331 3000 91.4% chr15 - 90130858 90131313 456 browser details YourSeq 152 984 1159 3000 92.1% chr5 + 100849863 100850027 165

Note: The 3000 bp section upstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

BLAT Search Results (down)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 - 24435579 24438578 3000 browser details YourSeq 197 2089 2476 3000 89.7% chr8 + 8933388 8933860 473 browser details YourSeq 189 2073 2437 3000 89.3% chr3 - 149058510 149059275 766 browser details YourSeq 187 2073 2435 3000 89.2% chr6 - 134970165 134970716 552 browser details YourSeq 176 1498 1718 3000 94.1% chr17 + 84894569 84894797 229 browser details YourSeq 175 2075 2475 3000 89.7% chr7 - 119027623 119028303 681 browser details YourSeq 174 2074 2428 3000 91.7% chr11 + 6699660 6884837 185178 browser details YourSeq 164 2099 2437 3000 90.7% chr4 - 57594761 57595353 593 browser details YourSeq 164 1474 1725 3000 96.2% chr9 + 57403677 57404129 453 browser details YourSeq 164 2078 2469 3000 84.5% chr2 + 178803946 178804242 297 browser details YourSeq 155 2073 2476 3000 84.4% chr3 - 149058621 149058899 279 browser details YourSeq 154 2075 2474 3000 82.1% chr17 - 65770480 65770786 307 browser details YourSeq 153 1514 1710 3000 90.8% chr2 + 86072627 86072813 187 browser details YourSeq 151 2074 2323 3000 89.7% chr8 + 8933576 8933875 300 browser details YourSeq 151 1496 1718 3000 86.8% chr1 + 52666908 52667086 179 browser details YourSeq 148 2136 2476 3000 90.2% chr9 - 98967827 98968471 645 browser details YourSeq 148 2101 2719 3000 81.7% chr15 + 27663002 27663271 270 browser details YourSeq 148 2075 2476 3000 82.3% chr11 + 115998400 115998623 224 browser details YourSeq 147 2078 2476 3000 85.8% chr9 + 42734633 42735014 382 browser details YourSeq 145 2144 2468 3000 92.4% chr7 + 101007444 101007794 351

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tcim transcriptional and immune response regulator [ Mus musculus (house mouse) ] Gene ID: 69068, updated on 26-Jun-2020

Gene summary

Official Symbol Tcim provided by MGI Official Full Name transcriptional and immune response regulator provided by MGI Primary source MGI:MGI:1916318 See related Ensembl:ENSMUSG00000056313 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as AW121743; AW321058; 1110065B09Rik; 1810011O10Rik Orthologs human all

Genomic context

Location: 8; 8 A2 See Tcim in Genome Data Viewer Exon count: 1

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (24437172..24438946, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (25548088..25549418, complement)

Chromosome 8 - NC_000074.6

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Transcript information: This gene has 1 transcript

Gene: Tcim ENSMUSG00000056313

Description transcriptional and immune response regulator [Source:MGI Symbol;Acc:MGI:1916318] Gene Synonyms 1810011O10Rik Location Chromosome 8: 24,437,180-24,438,984 reverse strand. GRCm38:CM001001.2 About this gene This gene has 1 transcript (splice variant), 276 orthologues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tcim-201 ENSMUST00000052622.5 1805 106aa ENSMUSP00000058631.4 Protein coding CCDS22194 Q9D915 TSL:NA GENCODE basic APPRIS P1

21.80 kb Forward strand 24.430Mb 24.435Mb 24.440Mb 24.445Mb Genes Gm26714-201 >antisense Gm44620-201 >TEC (Comprehensive set...

Contigs AC164300.3 > AC114602.16 > Genes (Comprehensive set... < Tcim-201protein coding

Regulatory Build

24.430Mb 24.435Mb 24.440Mb 24.445Mb Reverse strand 21.80 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000052622

< Tcim-201protein coding

Reverse strand 1.80 kb

ENSMUSP00000058... Pfam Arginine vasopressin-induced protein 1/transcriptional and immune response regulator

PANTHER Transcriptional and immune response regulator

All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Y K R

Variant Legend

synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 106

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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