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α1-Antitrypsin: Structure, Function, Physiology
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
There are at least four transcriptional initiation sites (Figure 1 A) in the 5′ flanking region of the human α1-AT gene.33,69 Three of these (– 2110, –2073, and –1892) are termed “macrophage specific” because these initiation sites are only used for constitutive α1-AT gene expression in cells of mononuclear phagocyte origin.69 The downstream transcriptional initiation site (+1) is termed “hepatocyte specific” because it is used by hepatocytes for constitutive α1-AT gene expression.33,69 This downstream transcriptional initiation site is also used for constitutive α1-AT gene expression by cells of enterocytic origin, whether crypt-like or having undergone differentiation to villous-like enterocytes.69 It is not yet known whether an alternative upstream translational initiation site at –354 is used in mononuclear phagocytes. This translational initiation site is followed by a short open-reading frame and termination codon.33 Furthermore, this translational codon is encoded within a context which is favorable for initiation of translation according to the Kozak consensus sequence principles.78,79 Other factors, such as a surrounding upstream flanking region which is heavily encumbered by secondary structure, may prevent efficient initiation at this site but still allow initiation of translation at the downstream site, which is known to precede the amino-terminal amino acid for the α1-AT protein produced in these cell types. There is another potential translational initiation codon at – 204, but according to the Kozak principles,78,79 it may be encumbered by the presence of intronic sequences within a short distance downstream.
Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets
Published in mAbs, 2018
Kathryn H. Ching, Ellen J. Collarini, Yasmina N. Abdiche, Daniel Bedinger, Darlene Pedersen, Shelley Izquierdo, Rian Harriman, Lei Zhu, Robert J. Etches, Marie-Cecile van de Lavoir, William D. Harriman, Philip A. Leighton
The human kappa construct, SynVK-CK, was identical to the SynVK-C construct previously described35 except for a modified Kozak consensus sequence in the functional V. The functional V region consists of a rearranged human VK3-15/JK4 gene and was obtained from screening a small library of VK3 family sequences from human PBMC RNA. For the VK pseudogenes, a diverse set of CDRs was selected from the naturally-occurring somatic human sequences found in the NCBI EST database, which was queried with the VK3-15 germline gene. Individual pseudogenes may consist of CDRs from different ESTs, and some framework diversity was also included in some of the pseudogenes. Framework 4 sequence was not included in the pseudogenes. For the human lambda construct, the functional V was swapped for a rearranged VL1-44 gene and VL-based pseudogenes. For the human heavy chain, the human functional VH was obtained from screening a small library of VH3 family sequences from human PBMC RNA. The selected V region is based on germline VH3-23 with a few somatic changes and is a rearranged V region consisting of V, D and JH4 elements. For the VH pseudogenes, two versions were made, and OmniChickens contained either one or the other. In construct SynVH-C (used in the BDNF and PGRN campaigns), a diverse set of CDRs was selected from the NCBI EST database, which was queried with the VH3-23 germline gene (Supplementary Fig. 2). Pseudogenes may have CDRs from different ESTs, some framework 1–3 diversity was included, but no framework 4 sequence was present in the pseudogenes. In construct SynVH-A7 (used in the cocktail immunization), the CDRs were composed of S, Y and W residues, with no changes in the frameworks relative to the functional VH (Supplementary Fig. 2). The pseudogenes were synthesized (Genewiz) with flanking 50 base pair spacers derived from the chicken heavy chain pseudogene locus and cloned into an array, all in the opposite orientation to the functional V. The IgH promoter region was amplified from chicken genomic DNA. The functional V and a short segment of the chicken J-Cμ intron were synthesized. These parts were assembled with a β-actin promoter and attB site for integration into the attP-neo present in the IgH KO.22